CA2201756C - Self-quenching fluorescence probe and method - Google Patents
Self-quenching fluorescence probe and method Download PDFInfo
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- CA2201756C CA2201756C CA002201756A CA2201756A CA2201756C CA 2201756 C CA2201756 C CA 2201756C CA 002201756 A CA002201756 A CA 002201756A CA 2201756 A CA2201756 A CA 2201756A CA 2201756 C CA2201756 C CA 2201756C
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
- C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
Abstract
An oligonucleotide probe is provided which includes a fluorescent reporter molecule and a quencher molecule capable of quenching the fluorescence of the reporter molecule. The oligonucleotide probe is constructed such that the probe exists in at least one single-stranded conformation when unhybridized where the quencher molecule is near enough to the reporter molecule to quench the fluorescence of the reporter molecule. The oligonucleotide probe also exists in at least one conformation when hybridized to a target polynucloetide where the quencher molecule is not positioned close enough to the reporter molecule to quench the fluorescence of the reporter molecule. By adopting these hybridized and unhybridized conformations, the reporter molecule and quencher molecule on the probe exhibit different fluorescence signal intensities when the probe is hybridized and unhybridized. As a result, it is possible to determine whether the probe is hybridized or unhybridized based on a change in the fluorescence intensity of the reporter molecule, the quencher molecule, or a combination thereof. In addition, because the probe can be designed such that the quencher molecule quenches the reporter molecule when the probe is not hybridized, the probe can be designed such that the reporter molecule exhibits limited fluorescence until the probe is either hybridized or digested.
designed such that the reporter molecule exhibits limited fluorescence until the probe is either hybridized or digested.
designed such that the reporter molecule exhibits limited fluorescence until the probe is either hybridized or digested.
Description
z~o~~~~
SELF-QUENCHING FLUORESCENCE PROBE AND METHOD
BACKGROUND OF THF TNVFNTION
Field of the Invention The invention relates generally to fluorescent probes which include a _ i fluorescent reporter molecule and a fluorescent quencher molecule. More specifically, the invention relates to fluorescent probes which include a .
fluorescent reporter molecule and a fluorescent quencher molecule which may be used in hybridization assays and in nucleic acid amplification reactions, especially polymerase chain reactions (PCR).
I)escrintion of Related Art Fluorescent reporter molecule - quencher molecule pairs have been incorporated onto oligonucleotide probes in order to monitor biological events based on the fluorescent reporter molecule and quencher molecule being separated or brought within a minimum quenching distance of each other. For example, probes have been developed where the intensity of the reporter molecule fluorescence increases due to the separation of the reporter molecule from the quencher molecule. Probes have also been developed which lose their fluorescence because the quencher molecule is brought into proximity with the reporter molecule. These reporter - quencher molecule pair probes have been used to monitor hybridization assays and nucleic acid amplification reactions, especially polymerase chain reactions (PCR), by monitoring either the appearance or disappearance of the fluorescence signal generated by the reporter molecule.
As used herein, a reporter molecule is a molecule capable of generating a fluorescence signal. A quencher molecule is a molecule capable of absorbing the fluorescence energy of an excited reporter molecule, thereby quenching the fluorescence signal that would otherwise be released from the excited reporter molecule. In order for a quencher molecule to quench an excited fluorophore, PGTlUS95114882-the quencher molecule must be within a minimum quenching distance of the excited reporter molecule at some time prior to the reporter molecule releasing the stored fluorescence energy.
Probes containing a reporter molecule - quencher molecule pair have been developed for hybridization assays where the probe forms a hairpin structure, i.e., where the probe hybridizes to itself to form a loop such that the quencher molecule is brought into proximity with the reporter molecule in the absence of a complementary nucleic acid sequence to prevent the formation of the hairpin structure. WO 90/03446; European Patent Application No. 0 601 889 A2. When a complementary target sequence is present, hybridization of the probe to the complementary target sequence disrupts the hairpin structure and causes the probe to adopt a conformation where the quencher molecule is no longer close enough to the reporter molecule to quench the reporter molecule.
As a result, the probes provide an increased fluorescent signal when hybridized to a target sequence than when unhybridized. Probes including a hairpin structure have the disadvantage that they can be difficult to design and may interfere with the hybridization of the probe to the target sequence.
Assays have also been developed for identifying the presence of a hairpin structure using two separate probes, one containing a reporter molecule and the other a quencher molecule. Mergney, et al., Nucleic Acids Research, 22:6 920-928 (1994). In these assays, the fluorescence signal of the reporter molecule decreases when hybridized to the target sequence due to the quencher molecule being brought into proximity with the reporter molecule.
One particularly important application for probes including a reporter -quencher molecule pair is their use in nucleic acid amplification reactions, such as polymerase chain reactions (PCR), to detect the presence and amplification of a target nucleic acid sequence. In general, nucleic acid amplification techniques have opened broad new approaches to genetic testing and DNA analysis.
Arnheim and Erlich, Ann. Rev. Biochem., 61: 131-156 (1992). PCR, in particular, has become a research tool of major importance with applications in, WO 96/15270 pCT/US95/14882 for example, cloning, analysis of genetic expression, DNA sequencing, genetic mapping and drug discovery. Arnheim and Erlich, Ann. Rev. Biochem., 61:
131-156 (1992); Gilliland et al., Proc. Natl. Acad. Sci., 87: 2725-2729 (1990);
Bevan et al., PCR Methods and Applications, 1: 222-228 (1992); Green et al., PCR Methods and Applications, 1: 77-90 (1991); Blackwell et al., Science, 250:
1104-1110 (1990).
The widespread applications of nucleic acid amplification techniques has driven the development of instrumentation for carrying out the amplification reactions under a variety of circumstances. Important design goals for such instrument development have included fine temperature control, minimization of sample-to-sample variability in mufti-sample thermal cycling, automation of pre- and post-reaction processing steps, high speed temperature cycling, minimization of sample volumes, real time measurement of amplification products and minimization of cross contamination, for example, due to "sample carryover". In particular, the design of instruments permitting amplification to be carried out in closed reaction chambers and monitored in real time would be highly desirable for preventing cross-contamination. Higuchi et al., Biotechnology, 10: 413-417 (1992) and 11: 1026-1030 (1993); and Holland et al., Proc. Natl. Acad. Sci., 88: 7276-7280 (1991). Clearly, the successful realization of such a design goal would be especially desirable in the analysis of diagnostic samples, where a high frequency of false positives and false negatives, for example caused by "sample carryover", would severely reduce the value of an amplification procedure. Moreover, real time monitoring of an amplification reaction permits far more accurate quantification of starting target DNA concentrations in multiple-target amplifications, as the relative values of close concentrations can be resolved by taking into account the history of the relative concentration values during the reaction. Real time monitoring also permits the efficiency of the amplification reaction to be evaluated, which can indicate whether reaction inhibitors are present in a sample.
SELF-QUENCHING FLUORESCENCE PROBE AND METHOD
BACKGROUND OF THF TNVFNTION
Field of the Invention The invention relates generally to fluorescent probes which include a _ i fluorescent reporter molecule and a fluorescent quencher molecule. More specifically, the invention relates to fluorescent probes which include a .
fluorescent reporter molecule and a fluorescent quencher molecule which may be used in hybridization assays and in nucleic acid amplification reactions, especially polymerase chain reactions (PCR).
I)escrintion of Related Art Fluorescent reporter molecule - quencher molecule pairs have been incorporated onto oligonucleotide probes in order to monitor biological events based on the fluorescent reporter molecule and quencher molecule being separated or brought within a minimum quenching distance of each other. For example, probes have been developed where the intensity of the reporter molecule fluorescence increases due to the separation of the reporter molecule from the quencher molecule. Probes have also been developed which lose their fluorescence because the quencher molecule is brought into proximity with the reporter molecule. These reporter - quencher molecule pair probes have been used to monitor hybridization assays and nucleic acid amplification reactions, especially polymerase chain reactions (PCR), by monitoring either the appearance or disappearance of the fluorescence signal generated by the reporter molecule.
As used herein, a reporter molecule is a molecule capable of generating a fluorescence signal. A quencher molecule is a molecule capable of absorbing the fluorescence energy of an excited reporter molecule, thereby quenching the fluorescence signal that would otherwise be released from the excited reporter molecule. In order for a quencher molecule to quench an excited fluorophore, PGTlUS95114882-the quencher molecule must be within a minimum quenching distance of the excited reporter molecule at some time prior to the reporter molecule releasing the stored fluorescence energy.
Probes containing a reporter molecule - quencher molecule pair have been developed for hybridization assays where the probe forms a hairpin structure, i.e., where the probe hybridizes to itself to form a loop such that the quencher molecule is brought into proximity with the reporter molecule in the absence of a complementary nucleic acid sequence to prevent the formation of the hairpin structure. WO 90/03446; European Patent Application No. 0 601 889 A2. When a complementary target sequence is present, hybridization of the probe to the complementary target sequence disrupts the hairpin structure and causes the probe to adopt a conformation where the quencher molecule is no longer close enough to the reporter molecule to quench the reporter molecule.
As a result, the probes provide an increased fluorescent signal when hybridized to a target sequence than when unhybridized. Probes including a hairpin structure have the disadvantage that they can be difficult to design and may interfere with the hybridization of the probe to the target sequence.
Assays have also been developed for identifying the presence of a hairpin structure using two separate probes, one containing a reporter molecule and the other a quencher molecule. Mergney, et al., Nucleic Acids Research, 22:6 920-928 (1994). In these assays, the fluorescence signal of the reporter molecule decreases when hybridized to the target sequence due to the quencher molecule being brought into proximity with the reporter molecule.
One particularly important application for probes including a reporter -quencher molecule pair is their use in nucleic acid amplification reactions, such as polymerase chain reactions (PCR), to detect the presence and amplification of a target nucleic acid sequence. In general, nucleic acid amplification techniques have opened broad new approaches to genetic testing and DNA analysis.
Arnheim and Erlich, Ann. Rev. Biochem., 61: 131-156 (1992). PCR, in particular, has become a research tool of major importance with applications in, WO 96/15270 pCT/US95/14882 for example, cloning, analysis of genetic expression, DNA sequencing, genetic mapping and drug discovery. Arnheim and Erlich, Ann. Rev. Biochem., 61:
131-156 (1992); Gilliland et al., Proc. Natl. Acad. Sci., 87: 2725-2729 (1990);
Bevan et al., PCR Methods and Applications, 1: 222-228 (1992); Green et al., PCR Methods and Applications, 1: 77-90 (1991); Blackwell et al., Science, 250:
1104-1110 (1990).
The widespread applications of nucleic acid amplification techniques has driven the development of instrumentation for carrying out the amplification reactions under a variety of circumstances. Important design goals for such instrument development have included fine temperature control, minimization of sample-to-sample variability in mufti-sample thermal cycling, automation of pre- and post-reaction processing steps, high speed temperature cycling, minimization of sample volumes, real time measurement of amplification products and minimization of cross contamination, for example, due to "sample carryover". In particular, the design of instruments permitting amplification to be carried out in closed reaction chambers and monitored in real time would be highly desirable for preventing cross-contamination. Higuchi et al., Biotechnology, 10: 413-417 (1992) and 11: 1026-1030 (1993); and Holland et al., Proc. Natl. Acad. Sci., 88: 7276-7280 (1991). Clearly, the successful realization of such a design goal would be especially desirable in the analysis of diagnostic samples, where a high frequency of false positives and false negatives, for example caused by "sample carryover", would severely reduce the value of an amplification procedure. Moreover, real time monitoring of an amplification reaction permits far more accurate quantification of starting target DNA concentrations in multiple-target amplifications, as the relative values of close concentrations can be resolved by taking into account the history of the relative concentration values during the reaction. Real time monitoring also permits the efficiency of the amplification reaction to be evaluated, which can indicate whether reaction inhibitors are present in a sample.
PCT/L1S95/1488~
Holland et al. (cited above), U.S. Patent No. 5,210,015 to Gelfand, et al.
and others have proposed fluorescence-based approaches to provide real time measurements of amplification products during PCR. Such approaches have either employed intercalating dyes (such as ethidium bromide) to indicate the amount of double-stranded DNA present, or they have employed probes _ containing fluorescence-quencher pairs (also referred to as the "Taq-Man"
approach) where the probe is cleaved during amplification to release a fluorescent molecule whose concentration is proportional to the amount of double-stranded DNA present. During amplification, the probe is digested by the nuclease activity of a polymerase when hybridized to the target sequence to cause the fluorescent molecule to be separated from the quencher molecule, thereby causing fluorescence from the reporter molecule to appear.
The Taq-Man approach, illustrated in Figure 1, uses an oligonucleotide probe containing a reporter molecule - quencher molecule pair that specifically anneals to a region of a target polynucleotide "downstream", i.e. in the direction of extension of primer binding sites. The reporter molecule and quencher molecule are positioned on the probe sufficiently close to each other such that whenever the reporter molecule is excited, the energy of the excited state nonradiatively transfers to the quencher molecule where it either dissipates nonradiatively or is emitted at a different emission frequency than that of the reporter molecule. During strand extension by a DNA polymerase, the probe anneals to the template where it is digested by the 5'->3' exonuclease activity of the polymerase. As a result of the probe being digested, the reporter molecule is effectively separated from the quencher molecule such that the quencher molecule is no longer close enough to the reporter molecule to quench the reporter molecule's fluorescence. Thus, as more and more probes are digested during amplification, the number of reporter molecules in solution increases, thus resulting in an increasing number of unquenched reporter molecules which produce a stronger and stronger fluorescent signal.
Holland et al. (cited above), U.S. Patent No. 5,210,015 to Gelfand, et al.
and others have proposed fluorescence-based approaches to provide real time measurements of amplification products during PCR. Such approaches have either employed intercalating dyes (such as ethidium bromide) to indicate the amount of double-stranded DNA present, or they have employed probes _ containing fluorescence-quencher pairs (also referred to as the "Taq-Man"
approach) where the probe is cleaved during amplification to release a fluorescent molecule whose concentration is proportional to the amount of double-stranded DNA present. During amplification, the probe is digested by the nuclease activity of a polymerase when hybridized to the target sequence to cause the fluorescent molecule to be separated from the quencher molecule, thereby causing fluorescence from the reporter molecule to appear.
The Taq-Man approach, illustrated in Figure 1, uses an oligonucleotide probe containing a reporter molecule - quencher molecule pair that specifically anneals to a region of a target polynucleotide "downstream", i.e. in the direction of extension of primer binding sites. The reporter molecule and quencher molecule are positioned on the probe sufficiently close to each other such that whenever the reporter molecule is excited, the energy of the excited state nonradiatively transfers to the quencher molecule where it either dissipates nonradiatively or is emitted at a different emission frequency than that of the reporter molecule. During strand extension by a DNA polymerase, the probe anneals to the template where it is digested by the 5'->3' exonuclease activity of the polymerase. As a result of the probe being digested, the reporter molecule is effectively separated from the quencher molecule such that the quencher molecule is no longer close enough to the reporter molecule to quench the reporter molecule's fluorescence. Thus, as more and more probes are digested during amplification, the number of reporter molecules in solution increases, thus resulting in an increasing number of unquenched reporter molecules which produce a stronger and stronger fluorescent signal.
wo 9sns2~o Three main factors influence the utility of reporter-quencher molecule pair probes in hybridization and amplification assays. The first factor is the effectiveness of the quencher molecule on the probe to quench the reporter molecule. This first factor, herein designated "RQ-", can be characterized by the ratio of the fluorescent emissions of the reporter molecule to the quencher molecule when the probe is not hybridized to a complementary polynucleotide.
That is, RQ' is the ratio of the fluorescent emissions of the reporter molecule to the fluorescence of the quencher molecule when the oligonucleotide probe is in a single-stranded state. Influences on the value of RQ' include, for example, the particular reporter and quencher molecules used, the spacing between the reporter and quencher molecules, nucleotide sequence-specific effects, and the degree of flexibility of structures, e.g., linkers, to which the reporter and quencher molecules are attached, and the presence of impurities. Wo et al., Anal. Biochem., 218: 1-13 (1994); and Clegg, Meth. Enzymol., 21 l: 353-388 (1992). A related quantity RQ+, refers to the ratio of fluorescent emissions of the reporter molecule to the quencher molecule when the oligonucleotide probe is hybridized to a complementary polynucleotide.
A second factor is the efficiency of the probe to hybridize to a complementary polynucleotide. This second factor depends on the probe's melting temperature, Tm, the presence of a secondary structure in the probe or target polynucleotide, the annealing temperature, and other reaction conditions.
A third factor is the efficiency with which the DNA polymerase 5'-~3' exonuclease activity cleaves the bound probe between the reporter molecule and quencher molecule. This efficiency depends on such factors as the proximity of the reporter or quencher to the 5' end of the probe, the "bulkiness" of the reporter or quencher, and the degree of complementarity between the probe and target polynucleotide. Lee et al., Nucleic Acids Research, 21: 3761-3766 ( 1993).
Since quenching depends on the physical proximity of the reporter molecule to the quencher molecule, it was previously assumed that the quencher ~~a~7~~
WO 96/15270 PGT/US95/1488J~
and reporter molecules must be attached to the probe such that the quencher molecule remains at all times within the maximum distance at which the quencher molecule can quench the reporter molecule, this distance generally being a separation of about 6-16 nucleotides. Lee et al. Nucleic Acids ' S Research, 21: 3761-3766 (1993); Mergny et al., Nucleic Acids Research 22:
920-928 (1994); Cardullo et al., Proc. Natl. Acad. Sci., 85: 8790-8794 (1988);
Clegg et al., Proc. Natl. Acad. Sci., 90: 2994-2998 (1993); and Ozaki et al., Nucleic Acids Research, 20: 5205-5214 (1992). This short separation between the reporter molecule and the quencher molecule is typically achieved by attaching one member of the reporter-quencher pair to the 3' or 5' end of the probe and the other member to an internal base 6-16 nucleotides away.
There are at least two significant disadvantages associated with attaching a reporter or quencher molecule to an internal base. Attaching a reporter or quencher molecule to an internal nucleotide typically involves more difficult chemistry than the chemistry required to attach the molecule to a terminal nucleotide. In addition, attachment of a reporter or quencher molecule to an internal nucleotide can adversely affect the hybridization efficiency of the probe. Ward et al., U. S. Patent 5,328,824; and Ozaki et al. Nucleic Acids Research, 20: 5205-5214 (1992).
A need currently exists for effective oligonucleotide probes containing a fluorescent reporter molecule and a quencher molecule for use in hybridization and nucleic acid amplification assays. Accordingly, a need exists for probes which exhibit distinguishable fluorescence characteristics when hybridized and not hybridized to a target nucleic acid sequence. A further need exists for probes where the reporter molecule and quencher molecule are positioned on the probe such that the quencher molecule can effectively quench the fluorescence of the reporter molecule. A further need exists for probes which are efficiently synthesized. Yet a further need exists for the reporter molecule and quencher molecule to be positionable on the probe such that the reporter and quencher molecules do not adversely impact the hybridization efficiency of probe. These 2~017~6 and further objectives are provided by the probes and methods of the present invention.
~IVIMARY OF THE INVENTION
The present invention relates to an oli~onucle~tirie "rr,hP .x~h;..h ;.".~.,ao~
o------------- t..a...... ..aaavaa laavluucJ
a fluorescent reporter molecule and a quencher molecule capable of quenching the fluorescence of the reporter molecule. According to the present invention, the oligonucleotide probe is constructed such that the probe exists in at least one single-stranded conformation when unhybridized where the quencher molecule is near enough to the reporter molecule to quench the fluorescence of the reporter molecule. The oligonucleotide probe also exists in at least one conformation when hybridized to a target polynucleotide where the quencher molecule is not positioned close enough to the reporter molecule to quench the fluorescence of the reporter molecule. By adopting these hybridized and unhybridized conformations, the reporter molecule and quencher molecule on the probe exhibit different fluorescence signal intensities when the probe is hybridized and unhybridized. As a result, it is possible to determine whether the probe is hybridized or unhybridized based on a change in the fluorescence intensity of the reporter molecule, the quencher molecule, or a combination thereof. In addition, because the probe can be designed such that the quencher molecule quenches the reporter molecule when the probe is not hybridized, the probe can be designed such that the reporter molecule exhibits limited fluorescence until the probe is either hybridized or digested.
According to the present invention, the fluorescence intensity of the reporter molecule is preferably greater than the fluorescence intensity of the quencher molecule when the probe is hybridized to the target polynucleotide.
The fluorescence intensity of the reporter molecule is more preferably at least about a factor of 3.5 greater than the fluorescence intensity of the quencher molecule when the probe is hybridized to the target polynucleotide.
The fluorescence intensity of the oligonucleotide probe hybridized to the target polynucleotide is also preferably at least about a factor of 6 greater than the fluorescence intensity of the oligonucleotide probe when not hybridized to the target polynucleotide.
The reporter molecule is preferably separated from the quencher _ molecule by at least about 15 nucleotides, more preferably at least about 18 nucleotides. The reporter molecule is preferably separated from the quencher molecule by between about 15 and 60 nucleotides, more preferably between about 18 and 30 nucleotides.
The reporter molecule is preferably attached to either the 3' or 5' terminal nucleotides of the probe. The quencher molecule is also preferably attached to either the 3' or 5' terminal nucleotides of the probe. More preferably, the reporter and quencher molecules are attached to the 3' and 5' or 5' and 3' terminal nucleotides of the probe respectively.
The reporter molecule is preferably a fluorescein dye and the quencher molecule is preferably a rhodamine dye.
In one embodiment, the oligonucleotide probe of the present invention is immobilized on a solid support. The oligonucleotide probe may be attached directly to the solid support, for example by attachment of the 3' or 5' terminal nucleotide of the probe to the solid support. More preferably, however, the probe is attached to the solid support by a linker. The linker serves to distance the probe from the solid support. The linker is most preferably at least 30 atoms in length, more preferably at least 50 atoms in length.
A wide variety of linkers are known in the art which may be used to attach the oligonucleotide probe to the solid support. The linker most preferably includes a functionalized polyethylene glycol because it does not significantly interfere with the hybridization of probe to the target oligonucleotide, is commercially available, soluble in both organic and aqueous media, easy to functionalize, and completely stable under oligonucleotide synthesis and post-synthesis conditions.
_8_ The linkages between the solid support, the linker and the probe are preferably not cleaved during removal of base protecting groups under basic conditions at high temperature. Examples of preferred linkages include carbamate and amide linkages.
The present invention also relates to the use of the oligonucleotide probe _ _ as a hybridization probe to detect target polynucleotides. Accordingly, the present invention relates to a hybridization assay for detecting the presence of a target polynucleotide in a sample. In one embodiment of the method, the hybridization probe is immobilized on a solid support.
According to the method, an oligonucleotide probe of the present invention is contacted with a sample of polynucleotides under conditions favorable for hybridization. The fluorescence signal of the reporter molecule before and after being contacted with the sample is compared. Since the reporter molecule on the probe exhibits a greater fluorescence signal when hybridized to a target sequence, an increase in the fluorescence signal after the probe is contacted with the sample indicates the hybridization of the probe to target sequences in the sample, thereby indicating the pressure of target sequences in the sample. Quantification of the change in fluorescence intensity as a result of the probe being contacted with the sample can be used to quantify the amount of target sequences present in the sample.
The present invention also relates to the use of the oligonucleotide probe for monitoring nucleic acid amplification. Accordingly, the present invention relates to a method for monitoring nucleic acid amplification by performing nucleic acid amplification on a target sequence using a nucleic acid polymerase having 5', 3' nuclease activity, a primer capable of hybridizing to the target sequence and an oligonucleotide probe according to the present invention which is capable of hybridizing to the target sequence 3' relative to the primer.
According to the method, the nucleic acid polymerase digests the oligonucleotide probe during amplification when it is hybridized to the target sequence, thereby separating the reporter molecule from the quencher molecule.
As the amplification is conducted, the fluorescence of the reporter molecule is monitored, the generation of fluorescence corresponding to the occurrence of nucleic acid amplification. Accordingly, the amount of amplification performed can be quantified based on the change in fluorescence observed. It is noted that the fluorescence of the quencher molecule may also be monitored, either separately or in combination with the reporter molecule to detect amplification.
According to an aspect of the invention, there is provided a method of nucleic acid amplification comprising:
performing nucleic acid amplification on a target polynucleotide using a nucleic acid polymerase having 5' to 3' nuclease activity, a primer capable of hybridizing to the target polynucleotide, and an oligonucleotide probe under amplification conditions such that the probe hybridizes to the target polynucleotide 3' relative to the primer and the probe does not hybridize with itself to form a hairpin structure, the oligonucleotide probe having at one end a fluorescent reporter and at the other end a quencher that quenches the fluorescence of the reporter molecule when both the fluorescent reporter and quencher are attached to the probe, under conditions such that digestion of the oligonucleotide probe by the polymerase during amplification is effective to separate the reporter from the quencher, whereby a fluorescence signal of the reporter is increased.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 illustrates a method for real-time monitoring nucleic acid amplification utilizing a probe which is degraded by the 5'~3' exonuclease activity of a nucleic acid polymerase.
Figure 2 illustrates a probe according to the present invention immobilized to a solid support in hybridized and unhybridized conformations.
- l0a -DETAILED DESCRIPTION
The present invention relates to an oligonucleotide probe which includes a fluorescent reporter molecule and a quencher molecule capable of quenching the fluorescence of the reporter molecule. According to the present invention, the oligonucleotide probe is constructed such that the probe exists in at least one single-stranded conformation when unhybridized where the quencher molecule is near enough to the reporter molecule to quench the fluorescence of the reporter molecule.
The oligonucleotide probe also exists in at least one conformation when hybridized to a target polynucleotide such that the quencher molecule is not positioned close enough to the reporter molecule to quench the fluorescence of the reporter molecule.
By adopting these hybridized and unhybridized conformations, the reporter molecule and quencher molecule on the probe exhibit different fluorescent signal intensities when the probe is hybridized and unhybridized. As a result, it is possible to determine whether the probe is hybridized or unhybridized based on a change in the fluorescence intensity of the reporter molecule, the quencher molecule, or a combination thereof. In addition, because the probe can be designed such that the quencher molecule quenches the reporter molecule when the probe is not hybridized, the probe can be designed such that the reporter molecule exhibits limited fluorescence unless the probe is either hybridized or digested.
According to the present invention, the fluorescence intensity of the reporter molecule is preferably greater than the fluorescence intensity of the quencher molecule when the probe is hybridized to the target polynucleotide.
The fluorescence intensity of the reporter molecule is more preferably at least about a factor of 3.5 greater than the fluorescence intensity of the quencher molecule when the probe is hybridized to the target polynucleotide.
The fluorescence intensity of the oligonucleotide probe hybridized to the target polynucleotide is also preferably at least about a factor of 6 greater than the fluorescence intensity of the oligonucleotide probe when not hybridized to the target polynucleotide.
The reporter molecule is preferably separated from the quencher molecule by at least about 15 nucleotides, more preferably at least about 18 nucleotides. The reporter molecule is preferably separated from the quencher molecule by between about 15 and 60 nucleotides, more preferably between about 18 and 30 nucleotides.
The reporter molecule is preferably attached to either the 3' or 5' terminal nucleotides of the probe. The quencher molecule is also preferably attached to either the 3' or 5' terminal nucleotides of the probe. More preferably, the reporter and quencher molecules are attached to the 3' and 5' or 5' and 3' terminal nucleotides of the probe respectively.
The reporter molecule is preferably a fluorescein dye and the quencher molecule is preferably a rhodamine dye.
In one embodiment, the oligonucleotide probe is attached to a solid support. As illustrated in Figure 2, when the probe is unhybridized, the probe is able to adopt at least one single-stranded conformation such that the quencher molecule is near enough to the reporter molecule to quench the fluorescence of WO 96/15270 2 ~ Q 17 5 6 pCT/US95114882~
the reporter molecule. As further illustrated in Figure 2, when the probe is hybridized to a target sequence, the probe adopts at least one conformation where the quencher molecule is not positioned close enough to the reporter , molecule to quench the fluorescence of the reporter molecule. As a result, the fluorescence intensity of the reporter molecule increases when the probe . ' hybridizes to a target sequence.
As illustrated in Figure 2, different probes may be attached to the solid support and may be used to simultaneously detect different target sequences in a sample. Reporter molecules having different fluorescence wavelengths can be used on the different probes, thus enabling hybridization to the different probes to be separately detected.
Examples of preferred types of solid supports for immobilization of the oligonucleotide probe include controlled pore glass, glass plates, polystyrene, avidin coated polystyrene beads, cellulose, nylon, acrylamide gel and activated dextran. CPG, glass plates and high cross-linked polystyrene. These solid supports are preferred for hybridization and diagnostic studies because of their chemical stability, ease of functionalization and well defined surface area.
Solid supports such as controlled pore glass (CPG, 500 A, 1000 ~) and non-swelling high cross-linked polystyrene (1000 t~) are particularly preferred in view of their compatibility with oligonucleotide synthesis.
The oligonucleotide probe may be attached to the solid support in a variety of manners. For example, the probe may be attached to the solid support by attachment of the 3' or 5' terminal nucleotide of the probe to the solid support. More preferably, however, the probe is attached to the solid support by a linker which serves to distance the probe from the solid support. The linker is most preferably at least 30 atoms in length, more preferably at least 50 atoms in length.
The length and chemical stability of linker between solid support and the first 3' unit of oligonucleotides play an important role in efficient synthesis and hybridization of support bound oligonucleotides. The linker arm should be zzo~~~s sufficiently long so that a high yield (>97%) can be achieved during automated synthesis. The required length of the linker will depend on the particular solid support used. For example, a six atom linker is generally sufficient to achieve a >97% yield during automated synthesis of oligonucleotides when high cross-linked polystyrene is used as the solid support. The linker arm is preferably at least 20 atoms long in order to attain a high yield (>97%) during automated synthesis when CPG is used as the solid support.
Hybridization of a probe immobilized to a solid support generally requires that the probe be separated from the solid support by at least 30 atoms, more preferably at least 50 atoms. In order to achieve this separation, the linker generally includes a spacer positioned between the linker and the 3' nucleoside.
For oligonucleotide synthesis, the linker arm is usually attached to the 3'-OH
of the 3' nucleoside by an ester linkage which can be cleaved with basic reagents to free the oligonucleotide from the solid support.
A wide variety of linkers are known in the art which may be used to attach the oligonucleotide probe to the solid support. The linker may be formed of any compound which does not significantly interfere with the hybridization of the target sequence to the probe attached to the solid support. The linker may be formed of a homopolymeric oligonucleotide which can be readily added on to the linker by automated synthesis. Alternatively, polymers such as functionalized polyethylene glycol can be used as the linker. Such polymers are preferred over homopolymeric oligonucleotides because they do not significantly interfere with the hybridization of probe to the target oligonucleotide. Polyethylene glycol is particularly preferred because it is commercially available, soluble in both organic and aqueous media, easy to functionalize, and completely stable under oligonucleotide synthesis and post-synthesis conditions.
The linkages between the solid support, the linker and the probe are preferably not cleaved during removal of base protecting groups under basic WO 96/15270 ~ 2 Q 1 ~ 5 6 PCT/US951i488~
conditions at high temperature. Examples of preferred linkages include carbamate and amide linkages.
The oligonucleotide probe of the present invention may be used as a hybridization probe to detect target polynucleotides. Accordingly, the present invention relates to a hybridization assay for detecting the presence of a target _ ' polynucleotide in a sample. According to the method, an oligonucleotide probe of the present invention is contacted with a sample of nucleic acids under conditions favorable for hybridization. The fluorescence signal of the reporter molecule is measured before and after being contacted with the sample. Since the reporter molecule on the probe exhibits a greater fluorescence signal when hybridized to a target sequence, an increase in the fluorescence signal after the probe is contacted with the sample indicates the hybridization of the probe to target sequences in the sample and hence the presence of target sequences in the sample. Further, by quantifying the change in fluorescence intensity as a result of the probe being contacted with the sample, the amount of target sequences in the sample can be quantified.
According to one embodiment of the method, the hybridization probe is immobilized on a solid support. The oligonucleotide probe is contacted with a sample of nucleic acids under conditions favorable for hybridization. The fluorescence signal of the reporter molecule is measured before and after being contacted with the sample. Since the reporter molecule on the probe exhibits a greater fluorescence signal when hybridized to a target sequence, an increase in the fluorescence signal after the probe is contacted with the sample indicates the hybridization of the probe to target sequences in the sample. Immobilization of the hybridization probe to the solid support enables the target sequence hybridized to the probe to be readily isolated from the sample. In later steps, the isolated target sequence may be separated from the solid support and processed ' (e.g., purified, amplified) according to methods well known in the art depending on the particular needs of the researcher.
The oligonucleotide probe of the present invention may also be used as a probe for monitoring nucleic acid amplification. Accordingly, the present invention relates to a method for monitoring nucleic acid-amplification using an oligonucleotide probe according to the present invention which is capable of hybridizing to the target sequence 3' relative to an ampIif cation primer.
According to the method, nucleic acid amplification is performed on a target polynucleotide using a nucleic acid polymerise having 5'- 3' nuclease activity, a primer capable of hybridizing to the target polynucleotide, and an oligonucleotide probe according to the present invention capable of hybridizing to the target polynucleotide 3' relative to the primer. During amplification, the nucleic acid polymerise digests the oligonucleotide probe when it is hybridized to the target sequence, thereby separating the reporter molecule from the quencher molecule. As the amplification is conducted, the fluorescence of the reporter molecule is monitored, the generation of fluorescence corresponding to 1 S the occurrence of nucleic acid amplification.
Use of a reporter-quencher pair probe generally in conjunction with the amplification of a target polynucleotide, for example, by PCR, e.g., is described in many references, such as Innis et al., editors, PCR Protocols (Academic Press, New York, 1989); Sambrook et al., Molecular Cloning, Second Edition (Cold Spring Harbor Laboratory, New York, 1989).
The binding site of the oligonucleotide probe is located between the PCR primers used to amplify the target polynucleotide.
Preferably, PCR is carried out using Taq DNA polymerise, e.g., AmplitaqTM
(Perkin-Elmer, Norwalk, CN), or an equivalent thermostable DNA polymerise, and the annealing temperature of the PCR is about ~-10°C below the melting temperature of the oligonucleotide probes employed.
Use of an oligonucleotide probe according to the present invention for monitoring nucleic acid amplification provides several advantages over the use of prior art reporter-quencher pair probes. For e:cample, prior art probes required that the reporter and quencher molecules be positioned on the probe - I~-such that the quencher molecule remained within a minimum quenching distance of the reporter molecule. However, by realizing that the probe need only be designed such that the probe be able to adopt a conformation where the quencher molecule is within a minimum quenching distance of the reporter molecule, a far wider array of probes are enabled. For example, dually labelled probes having the reporter and quencher molecules at the 5' and 3' ends can be designed. Such probes are far easier to synthesize than probes where the reporter molecule or the quencher molecule is attached to an internal nucleotide.
Positioning of the reporter and quencher molecules on terminal nucleotides also enhances the hybridization efficiency of the probes.
As used in this application, the term "oligonucleotide", includes linear oligomers of natural or modified monomers or linkages, including deoxyribonucleosides, ribonucleosides, and the like; capable of specifically binding a target polynucleotide by way of a regular pattern of monomer-to-monomer interactions, such as Watson-Crick type of basepairing, or the like.
Usually monomers are linked by phosphodiester bonds or analogs thereof to form oligonucleotides ranging in size from a few monomeric units, e.g., 3-4, to several tens of monomeric units. Whenever an oligonucleotide is represented by a sequence of letters, such as "ATGCCTG", it will be understood that the nucleotides are in 5' --~ 3' order from left to right and that "A" denotes deoxyadenosine, "C" denotes deoxycytidine, "G" denotes deoxyguanosine, and "T" denotes thymidine, unless otherwise noted. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoranilidate, phosphoramidate, and the like. Generally, oligonucleotide probes of the invention will have a sufficient number of phosphodiester linkages adjacent to its 5' end so that the 5' -~ 3' exonuclease activity employed can efficiently degrade the bound probe to separate the reporter and quencher molecules.
"Perfectly matched" in reference to a duplex means that the poly- or oligonucleotide strands making up the duplex form a double-stranded structure with one other such that every nucleotide in each strand undergoes Watson-22Q175~
Crick basepairing with a nucleotide in the other strand. The term also comprehends the pairing of nucleoside analogs, such as deoxyinosine, nucleosides with 2-aminopurine bases, and the like, that may be employed.
Conversely, a "mismatch" in a duplex between a target polynucleotide and an oligonucleotide probe or primer means that a pair of nucleotides in the duplex fails to undergo Watson-Crick bonding.
As used in the application, "nucleoside" includes the natural nucleosides, including 2'-deoxy and 2'-hydroxyl forms, e.g., as described in Kornberg and Baker, DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992). "Analogs" in reference to nucleosides includes synthetic nucleosides having modified base moieties and/or modified sugar moieties, e.g., described by Scheit, Nucleotide Analogs (John Wiley, New York, 1980); Uhlman and Peyman, Chemical Reviews, 90: 543-584 (1990), or the like, with the only proviso that they are capable of specific hybridization. Such analogs include synthetic nucleosides designed to enhance binding properties, reduce degeneracy, increase specificity, and the like.
Oligonucleotide probes of the invention can be synthesized by a number of approaches, e.g., Ozaki et al., Nucleic Acids Research, 20: 5205-5214 (1992); Agrawal et al., Nucleic Acids Research, 18: 5419-5423 (1990); or the like. The oligonucleotide probes of the invention are conveniently synthesized on an automated DNA synthesizer, e.g., an Applied Biosystems, Inc. (Foster City, California) model 392 or 394 DNA/RNA Synthesizer, using standard chemistries, such as phosphoramidite chemistry, e.g., disclosed in the following references: Beaucage and Iyer, Tetrahedron, 48: 2223-2311 (1992); Molko et al., U. S. Patent 4,980,460; Koster et al., U. S. Patent 4,725,677; Caruthers et al., U. S. Patents 4,415,732; 4,458,066; and 4,973,679; and the like.
Alternative chemistries, e.g., resulting in non-natural backbone groups, such as phosphorothioate, phosphoramidate, and the like, may also be employed provided that the hybridization efficiencies of the resulting oligonucleotides WO 96/15270 PCT/US95/14882~
and/or cleavage efficiency of the exonuclease employed are not adversely affected.
Preferably, the oligonucleotide probe is in the range of 15-60 nucleotides in length. More preferably, the oligonucleotide probe is in the range of 18-30 nucleotides in length. The precise sequence and length of an oligonucleotide probe of the invention depends in part on the nature of the target polynucleotide to which it binds. The binding location and length may be varied to achieve appropriate annealing and melting properties for a particular embodiment.
Guidance for making such design choices can be found in many of the above-cited references describing the "Taq-man" type of assays.
Preferably, the 3' terminal nucleotide of the oligonucleotide probe is blocked or rendered incapable of extension by a nucleic acid polymerase. Such blocking is conveniently carried out by the attachment of a reporter or quencher molecule to the terminal 3' carbon of the oligonucleotide probe by a linking moiety.
Preferably, reporter molecules are fluorescent organic dyes derivatized for attachment to the terminal 3' carbon or terminal 5' carbon of the probe via a linking moiety. Preferably, quencher molecules are also organic dyes, which may or may not be fluorescent, depending on the embodiment of the invention.
~ For example, in a preferred embodiment of the invention, the quencher molecule is fluorescent. Generally whether the quencher molecule is fluorescent or simply releases the transferred energy from the reporter by non-radiative decay, the absorption band of the quencher should substantially overlap the fluorescent emission band of the reporter molecule. Non-fluorescent quencher molecules that absorb energy from excited reporter molecules, but which do not release the energy radiatively, are referred to in the application as chromogenic molecules.
There is a great deal of practical guidance available in the literature for selecting appropriate reporter-quencher pairs for particular probes, as exemplified by the following references: Clegg (cited above); Wu et al. (cited above); Pesce et al., editors, Fluorescence Spectroscopy (Marcel Dekker, New York, 1971); White et al., Fluorescence Analysis: A Practical Approach (Marcel Dekker, New York, 1970); and the like. The literature also includes references providing exhaustive lists of fluorescent and chromogenic molecules and their relevant optical properties for choosing reporter-quencher pairs, e.g., Berlman, Handbook of Fluorescence Spectra of Aromatic Molecules, 2nd Edition (Academic Press, New York, 1971); Griffiths, Colour and Constitution of Organic Molecules (Academic Press, New York, 1976); Bishop, editor, Indicators (Pergamon Press, Oxford, 1972); Haugland, Handbook of Fluorescent Probes and Research Chemicals (Molecular Probes, Eugene, 1992) Pringsheim, Fluorescence and Phosphorescence (Interscience Publishers, New York, 1949); and the like. Further, there is extensive guidance in the literature for derivatizing reporter and quencher molecules for covalent attachment via common reactive groups that can be added to an oligonucleotide, as exemplified by the following references: Haugland (cited above); Ullman et al., U. S.
Patent 3,996,345; Khanna et al., U. S. Patent 4,351,760; and the like.
Exemplary reporter-quencher pairs may be selected from xanthene dyes, including fluoresceins, and rhodamine dyes. Many suitable forms of these compounds are widely available commercially with substituents on their phenyl moieties which can be used as the site for bonding or as the bonding functionality for attachment to an oligonucleotide. Another group of fluorescent compounds are the naphthylamines, having an amino group in the alpha or beta position. Included among such naphthylamino compounds are 1-dimethylaminonaphthyl-S-sulfonate, 1-anilino-8-naphthalene sulfonate and 2-p-touidinyl-6-naphthalene sulfonate. Other dyes include 3-phenyl-7-isocyanatocoumarin, acridines, such as 9-isothiocyanatoacridine and acridine orange; N-(p-(2-benzoxazolyl)phenyl)maleimide; benzoxadiazoles, stilbenes, pyrenes, and the like.
Preferably, reporter and quencher molecules are selected from fluorescein and rhodamine dyes. These dyes and appropriate linking methodologies for attachment to oligonucleotides are described in many references, e.g., Khanna et al. (cited above); Marshall, Histochemical J., 7:
303 (1975); Menchen et al., U. S. Patent 5,188,934; Mencl~en et al., European Patent Application 87310256.0; and Bergot et al., International Application PCT/US90/05565.
There are many linking moieties and methodologies for attaching reporter or quencher molecules to the 5' or 3' termini of oIigonucleotides, as exemplified by the following references: Eckstein, editor, Oligonucleotides and Analogues: A Practical Approach (IRL Press, Oxford, 1991); Zuckerman et al., Nucleic Acids Research, 15: 5305-5321 (1987) (3' thiol group on oligonucleotide); Sharma et al., Nucleic Acids Research, 19: 3019 (1991) (3' sulflnydryl); Giusti et al., PCR Methods and Applications, 2: 223-227 (1993) and Fung et al., U. S. Patent 4, 757,141 (5' phosphoamino group via AminolinkTM II available from Applied Biosystems, Foster City, CA) Stabinsky, U. S. Patent 4,739,044 (3' aminoalkylphosphoryl group); Agrawal et al., Tetrahedron Letters, 31: 1543-1546 (1990) (attachment via phosphoramidate linkages); Sproat et al., Nucleic Acids Research, 15: 4837 (1987) (5' mercapto group); Nelson et al., Nucleic Acids Research, 17: 7187-7194 ( 1989) (3' amino group); and the like.
Preferably, commercially available linking moieties are employed that can be attached to an oligonucleotide during synthesis; e.g., available from Clontech Laboratories (Palo Alto, CA).
Rhodamine and fluorescein dyes are also conveniently attached to the 5' hydroxyl of an oIigonucleotide at the conclusion of solid phase synthesis by way of dyes derivatized with a phosphoramidite moiety, e.g., Woo et al., U. S.
Patent 5,231, 191; and Hobbs, Jr., U. S. Patent 4,997,928.
The following examples set forth probes and methods for using the probes according to the present invention. It is understood that the specific probes, probe constructs and steps of the methods described in these examples are,not intended to be limiting. Further objectives and advantages of the present invention other than those set forth above will become apparent from the examples which are not intended to limit the scope of the present invention.
EXAMPLES
1. SmtheSis n Oli~onucleotide Probes The following example describes the synthesis of the oligonucleotides shown in Table 1. Linker arm nucleotide ("LAN") phosphoramidite was obtained from Glen Research. Standard DNA phosphoramidites, 6-carboxyfluorescein ("6-FAM") phosphoramidite, 6- ...
carboxytetramethyIrhodamine succinimidyl ester ("TAMRA NHS ester"), and PhosphalinkTM for attaching a 3' blocking phosphate were obtained from Perkin-Elmer, Applied Biosystems Division. Oligonucleotide synthesis was performed on a model 394 DNA Synthesizer (Applied Biosystems). Primer and complement oIigonucleotides were purified using OIigo Purification Cartridges (Applied Biosystems).. Doubly labeled probes were synthesized with 6-FAM-labeled phosphoramidite at the 5' end, LAN replacing one of the T's in the oligonucIeotide sequence, and PhosphalinkTM at the 3' end. Following deprotection and ethanol precipitation, TAMRA NHS ester was coupled to the LAN-containing oligonucleotide in 250 mM Na-bicarbonate buffer (pH 9.0) at room temperature. Unreacted dye was removed by passage over a PD-10 Sephadex column. Finally, the doubly labeled probe was purified by preparative HPLC using standard protocols. Below, probes are named by designating the sequence from Table I and the position of the LAN-TAMRA
moiety. For example, probe AI-7 has sequence of AI with LAN-TAMR.A at nucleoside position 7 from the 5' end.
*Trademark WO 96/15270 ~ 2 Q 1 7 5 ~ pGT/US95/14882 Table 1. Sequences of oligonucleotides Name Type Sequence F 119 primer ACCCACAGGAACTGATCACCACTC
[SEQ. ID. No.: 1 ]
8119 primer ATGTCGCGTTCCGGCTGACGTTCTGC
[SEQ. ID. No.: 2]
P2 probe TCGCAT~ACTGATCGTTGCCAACCAGTp [SEQ. ID. No.: 3]
P2C complement GTACTGGTTGGCAACGATCAGTAATGCGATG
[SEQ. ID. No.: 4] .
PS probe CGGATTTGCTGGTATCTATGACAAGGATp [SEQ. ID. No.: 5]
PSC complement TTCATCCTTGTCATAGATACCAGCAAATCCG
[SEQ. ID. No.: 6]
AFP primer TCACCCACACTGTGCCCATCTACGA
[SEQ. ID. No.: 7]
ARP primer CAGCGGAACCGCTCATTGCCAATGG
[SEQ. ID. No.: 8]
Al probe A_TGCCC_TCCCCCA_TGCCA_TCC_TGCGTp [SEQ. ID. No.: 9]
A1C complement AGACGCAGGATGGCATGGGGGAGGGCATAC
[SEQ. ID. No.: 10]
A3 probe CGCCCTGGACTTCGAGCAAGAGAT~
[SEQ. ID. No.: 11]
A3C complement CCATCTCTTGCTCGAAGTCCAGGGCGAC
[SEQ. ID. No.: 12]
Gl probe CAAGCTTCCCGTTCTCAGCCT
[SEQ. ID. No.: 13]
G1C complement ACCGTCAAGGCTGAGAACGGGAAGCTTGTC
[SEQ. ID. No.: 14]
22n1756 Table 2.
O
NHFmoc HO
HOBT, HBTU, DIPEA, DL-Homoserine O
H~ NHFmoc N
DMT-Cl, DMAP, pyr O
H~ NHFmoc N
Polyethylene glycol) bis(2-aminoethyl ether), HOBT, HBTU, DIPEA, DMF
o H~ NHFmoc N
H
DMTO N
~ PEG-NH2 Succinic anhydride, DMAP, E~N,CH2C12 O
H~ HFmoc N
N/H /H O
DMT ~ ~ PEG-N H
O
WO 96/15270 pCT/LTSg5114882 CG
x.-z ~ x.-z i.-O ~
O
N N
x x U U
_ x Z
U
U
o O- U
x I I
x-z- U-= x U
U Z U
a O- U
O~. U ~
a a Z x_ Z .Q x- Z ~ ~' V
m ~ Z a a c I x i GL..'~ v ~ ~ m ~ .;
m x z a =~ Z suesn $
I ~ _ O-U ~
= O-U
~ a 'e a V ~ V 'm a N E' ~ ~ ~
' O-. sa.~ O= D
y x~Z _ x-z N
x ~I
r V
~/
v 2~0 ~~5s WO 96/15270 PCTlUS95/14882 L °
N
U IN
V ~ U
O=U Z I O=U Z
I
Z-Z-U-U c Z-Z- i U ._°.
o= I g O-U
z-z ~ z-z I
m 0. m c I a o D
O= ~ O-U
U
h N
N N O
a°
O=U ~ O=U
N z ( o z z-z ~ r~ z-z o a a v~
E= V ~ = V m m ..z a O=U = C O=U N ~ O=U x a Q ea ~
o -.
U O U ,_ U
a a>
O=U O=U ~ O=U
Z-Z Z-Z =-2 = U = = U = = U
U ( U U ~ U U I U
O-tn-O O-~n-O O-VJ-O
O O O
:. ~. ;r :r ::
a~
.o :a L
WO 96/15270 ~ pGT/U895/1488~
2. Synthesis of Oli~onucleotide Probes Attached To A Solid Su_pnort Both high cross-linked polystyrene (1000 A) and controlled pore glass (CPG) (500 A) are used as solid support matrices. The functionalization of a spacer (compound S 5) is illustrated in Table 2. The attachment of the spacer to polystyrene and CPG
supports, and the labelling of the solid supports with TAMRA dye is shown in Tables 3 and 4 respectively.
Table 2 illustrates a reaction scheme for the synthesis of a spacer, compound 5, which is used to derivatize CPG and polystyrene supports. As shown in Table 2, N-Fmoc-e-aminocaproic acid was reacted with DL-homoserine in presence of HOBT/HBTU/DIPEA (Knorr, et al., Tetrahedron Lett. 1989, 30, 1927) in DMF to give compound 2 in 65% yield. Compound 2 was reacted with dimethoxytrityl chloride in presence of DMAP in pyridine to give compound 3 in 72% yield after chromatography.
Treatment of compound 3 with a large excess of PEG-diamine (Buckmann, et al., Biotech. Appl. Biochem 1987, 9, 258) in presence of HOBT/HBTU/DIPEA in DMF
afforded amine 4 in 60% yield. The amine 4 was then converted to succinate 5 by treating amine 4 with succinic anhydride/Et3N/DMAP in CHZCIz in 90% yield. The succinate 5 was then attached to polystyrene and CPG support as illustrated in Tables 3 and 4 respectively without further purification.
As illustrated in Tables 3 and 4, succinate 5 was separately reacted with polystyrene and CPG support in presence of HOBT/HBTU/DIPEA in DMF to provide functionalized support 6 (5 ~mol/g loading) and functionalized support 8 (15 ~mol/g loading) respectively. The Fmoc group was removed from support bound spacer by treating supports 6 and 8 with 20% piperidine in DMF (Fields, et al., JPeptide Res.
1990, 35, 161) to give amine which was reacted with TAMRA NHS ester to give TAMRA labeled supports 7 and 9 respectively. The polystyrene and CPG supports showed a final loading of 4.8 ~.mol/g and 14 ~,mol/g respectively by trityl cation assay.
Double labeled Taqman probe was synthesized using both TAMRA labeled supports 7 and 9, FastPhoramidites (User Bulletin Number 85, Perkin Elmer Corporation 1994) and FAM phosphoramidite (User Bulletin Number 78, Perkin Elmer Corporation zzo ~7~s 1994) in 40 nanomol scale. The support bound oligonucleotides were deprotected by treating with MeOHa-BuNH2:H20 (1:1:2) at 65 °C for 3 hours (Woo, et al., U.S. Patent No. 5,231,191). Liquid was removed and the support containing probes were washed with H20:MeOH (3:1) and MeOH. The support was then dried under vacuum and used in a hybridization assay. _ Experimental:
Compound 2: N,N Diisopropylethylamine (l.lg, 1.48 mL, 8.52 mmol), 1-hydroxybenzotriazol (420 mg, 3.1 mmol) and (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (1.17 g, 3.1 mmol) were added to a stirred solution of Nfmoc-e-aminocaproic acid (1 g, 2.84 mmol) in DMF (30 mL) at room temperature. After 15 min DL-homoserine (1.35 g, 11.36 mmol) was added to the reaction mixture. After 3 hours, DMF was removed under reduced pressure. The residue was dissolved in CHC13 (100 mL) and washed with 5% aqueous HCl (2 X 50 mL).
The organic layer was dried over MgS04 and evaporated to give a thick oil which was trituated with ether to give a colorless solid (840 mg, 65%). The product was left under high vacuum for 24 hours and used in the next step without fiufiher purification.
Compound 3: 4,4'-Dimethoxytrityl chloride (484mg, 1.43 mmol) and 4-dimethyaminopyridine (25mg, 0.2 mmol) were added to a stirred solution of compound 2 (500mg, 1.1 mmol) in pyridine (15 mL) at room temperature under nitrogen atmosphere.
After 14 hours, pyridine was removed and the residue was dissolved in CHCl3 (70 mL).
The organic layer was extracted with 5% aqueous citric acid (1 X 50 mL), H20 (1 X 50 mL) and saturated brine (1 X 50 mL). The organic layer was dried over MgS04 and evaporated to give a yellow foam. The product was purified by a silica gel column eluting with CHCl3-MeOH gradient (0-10% MeOH). The appropriate fractions were combined and evaporated to give Compound 3 as a colorless foam (600 mg, 72%).
_ Compound 4: Polyethylene glycol) bis(2-aminoethyl ether) (3.16 g, 5.3 mmol), N, N
diisopropylethylamine (205 mg, 0.27 mL, 1.59 mmol), 1-hydroxybenzotriazol (78 mg, wo 96ns2~o PCTlITS95/14882~
0.58 mmol) and (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluonium hexafluorophosphate (220 mg, 0.58 mmol) were added to a stirred solution of compound 3 (400 mg, 0.53 mmol) in DMF (10 mL) at room temperature. The reaction mixture was _ stirred at room temperature for 3 hours. DMF was removed under reduced pressure and, the residue was dissolved in CHCl3 (70 mL) and washed with H20 (1 X 50 mL) and saturated brine (2 X 50 mL). The organic layer was dried over MgS04 and evaporated to give a thick oil. Compound 4 was purified by a silica gel column eluting with a CHC13-MeOH gradient (5-15% MeOH) as a colorless glass (423 mg, 60%).
Compound 5: Succinic anhydride (22 mg, 0.22 mmol), Et3N (23 mg, 0.31 ~L, 0.22 mmol), 4-dimethylaminopyridine (14 mg, 0.11 mmol) were added to a solution of compound 4 (300 mg, 0.22 mmol) in CHzCIz (15 mL). The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was diluted with CHC13 (30 mL) and washed with 5% aqueous citric acid (1 X 50 mL) and saturated brine (2 X 50 mL).
The organic layer was dried over MgS04 and evaporated to a colorless foam (284 mg, 90%) which was used for derivatization of the solid support without further purification.
Derivatization of Polystyrene support with TAMRA dye: High cross linked polystyrene (1000 A, 10 ~,mol/g amine loading, lg, 10 ~mol), was treated with compound 5 (17 mg, 12 p,mol, .1-hydroxybenzotriazol (1.8 mg, 12 ~.mol), (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluonium hexafluorophosphate (4.8 mg, 12 ~mol), N,N diisopropylethylamine (6 ~L, 30 ~,mol) in DMF (10 mL) on a wrist action shaker for 4 hours at room temperature. The support was washed with DMF (3 X 10 mL), (2 X 10 mL) and CHZCIz (1 X 10 mL) and dried under high vacuum overnight. The ninhydrin assay showed 1 ~mol/g amine left. The trityl cation assay gave 5 ~mol/g loading of compound 5. The support was capped with acetic anhydride/lutidine in THF
(10% solution, 5 mL) and 1-methylimidazol in THF (16% solution, 5 mL) for 2 hours at room temperature. The support was washed with CH3CN (3 X 10 mL) and CHZCI, (1 X
10 mL). The support was treated with 20% piperidine in DMF (3 X 10 mL) to remove the Fmoc protecting group. The removal of the Fmoc group was monitored by zz~~~~s measuring UV of the solution at 302 nm. The support was washed with DMF (3 X
mL) and, then treated with TAMItA NHS ester (15 mg, 27 ~,mol) and Et3N (50 ~mol) in DMF (10 mL) for 42 hours on a shaker. The support was washed with DMF (3 X 10 mL) CH3CN (2 X 10 mL) and CH2Clz (1 X 10 mL) and dried under high vacuum for S hours.- Ninhydrin test showed less than 0.5 ~,mol/g amine left. The support was capped with acetic anhydride/lutidine in THF (10% solution, 5 mL) and 1-methylimidazol in THF (16% solution, 5 mL) for 1 hour and then washed with CH3CN (3 X 10 mL), CH2Cl2 (2 X 10 mL) and dried under high vacuum for 24 hour. The trityl cation assay showed a final loading of 4.8 ~mol/g.
Derivatization of CPG support with TAMItA dye: A mixture of CPG (500 A, 40 pmol/g amine loading, 500 mg, 20 ~,mol), compound 5 (31 mg, 22 ~mol), 1-hydroxybenzotriazol (5.9 mg, 22 ~,mol), (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexaflurophosphate (8.4 mg, 22 ~.mol), N,N
diisopropylethylamine (10.4 ~,L, 60 ~,mol) in DMF (10 mL) was shaken on a wrist action shaker for 4 hours at room temperature. The support was washed with DMF (3 X 10 mL), CH3CN (2 X 10 mL) and CHZC12 (1 X 10 mL) and dried under high vacuum overnight. The ninhydrin assay showed 4 ~,mol/g amine left. The trityl assay gave 15 ~,mol/g loading of compound 5 on CPG support. The support was capped with acetic anhydride/lutidine in THF (10% solution, 5 mL) and 1-methylimidazol in THF (16% solution, 5 mL) for hours at room temperature. The support was washed with CH3CN (3 X 10 mL) and CH.,C12 (1 X 10 mL). The support was treated with 20% piperidine in DMF (3 X
10 mL) to remove the Fmoc protecting group. Removal of the Fmoc group was monitored by measuring UV of the solution at 302 nm. The support was washed with DMF (3 X
mL). The support was then treated with TAMRA NHS ester (25 mg, 45 ~mol) and Et3N
(90 ~mol) in DMF (5 mL) for 42 hours on a shaker. The support was washed with DMF
(3 X 10 mL), CH3CN (2 X 10 mL) and CHZC12 (1 X 10 mL) and dried under high vacuum for 24 hours. Ninhydrin test showed less than 1 ~mol/g amine left. The support - was capped with acetic anhydride/lutidine in THF (10% solution, 5 mL) and 1-methylimidazol in THF (16% solution, 5 mL) for 1 hour and then washed with (3 X I 0 mL), CH,CIz (2 X 10 mL) and dried under high vacuum for 24 hours. The-trityl cation assay showed a final loading of 14 ~mol/g.
Synthesis of FAM and TAMRA Doubled Labeled Probes: Doubled dye labeled probes were synthesized by using TAMR.A labelled supports 7 and 9, DNA
FastPhosphoramidite and FAM amidite in 40 nmol scale. After completion of synthesis, supports containing probes were transferred to 4 mL glass vials and treated with a mixtl.tre of MeOHa-BuNHz:H20 (l : I :2) at 65 °C for 3 hours. Liquid was removed by a syringe and the support was washed with H~O:MeOH (3:1) and MeOH. The support was dried under vacuum and used in the hybridization assay.
3. Hybridization Ascay Usinp~jlgonucleotide Probe A 295 basepair segment of exon 3 of human beta-actin gene (nucleotides 2141-243 as disclosed in Nakajima-Iijima, S., Proc. Natl. Acad. ci U~ 82:
6133-6137 (1985) can be amplified using 50 ul reactions that contain 10 mM
Tris-HCl (pH 8.3), 50 mM KCI, 4 mM MgClz, 300 nM primer AFP [SEQ. LD. No. 7J, 300 nM
primer biotin-ARP [SEQ. LD. No. 8 with biotin attached to the 5' endJ, 200 ~eM
dATP, 200 ~M dCTP, 200 p.M dGTP, 200 p.M TTP, and 1.25 units AmpliTaq (Perkin-EImer).
The reactions are performed with (+ template) or without (no template) 20 ng human genomic DNA.
After thermal cycling at 50 °C (2 min); 95 °C (10 min); and 40 cycles of 95 °C (20 sec) followed by 60 °C (1 min), each sample is diluted by adding 200 p1 Hybridization Buffer (SX SSC, 8% (v/v) formamide, 8% (v/v) Triton X- 100). The resulting samples are transferred to a streptavidin-coated 96-well microtiter plate (Xenopore Corp., Saddle Brook, NJ) and incubated at 37 °C for 30 min in order to capture the amplified beta-actin DNA segment. Each well is then washed with 330 pl phosphate buffered saline/0.05%
TWEEN-20. Any unbiotinylated DNA strands are removed by adding 100 ~I O.I M
NaOH / 1 mM EDTA, incubating at room temperature for ~ min, and washine with ul phosphate buffered saline/0.0~% TWEEN-20. 50 ul of Hybridization Buffer containing 100 nVI ofprobe A1-26 [SEQ. LD. No. 9, nucleotides 1-26 (.~1-26), labeled *Trademark WO 96!15270 PCT/US95/14882 with reporter FAM and quencher TAMRA) is then added and incubate at 37 °C for 30 mm.
Fluorescence is then measured at 518 nm and 582 nniusing a Perkin-Elmer TaqMan LS-SOB System. The ~RQ is then calculated as described in Example 5.
The magnitude of oRQ indicates the level of hybridization of the A1-26 probe and thus is a measure of the amount of amplified beta-actin DNA segment captured in each well.
4. Hybridization Assay Using Qlieonucleotide Probe Attached To Solid SuRport Three probe/solid support combinations were examined: A1-PS: A1 [SEQ. LD.
No. 9] attached to polystyrene support; A1-CPG: Al [SEQ. LD. No. 9] attached tb glass support; and Gl-PS: G1 [SEQ. LD. No. 13] attached to polystyrene support.
All three probes have FAM attached to the 5' end of the sequence and TAMRA
attached to the 3' end. No template reactions were prepared by suspending each probe/solid support sample in 50 p1 1X PCR Buffer (10 mM Tris-HCl (pH 8.3), 50 mM
KCI, 3.5 mM MgClz). For plus template reactions, A 1-PS and A 1-CPG were suspended in 50 ~l 1X PCR Buffer+ 1 ~M A1C; G1-PS was suspended in 50 ul 1X PCR Buffer+
1 ~M G1C.
Reactions were incubated at 95 °C for 1 min, then allowed to cool slowly to room temperature. A portion of each suspension was placed on a microscope slide.
Each sample was excited with 488 nm light and a fluorescence image was captured on a CCD
array using either a 518 nm or 583 nm interference filter. The images were analyzed by Ending a peak pixel value on the 518 nm image and then finding the 583 nm value for 2~ the same pixel. Pixel values were corrected by subtracting the background readings observed with buffer. Table ~ gives the results of fluorescence measurements of the indicated probes.
*Trademark WO 96/15270 2 2 ~ 1 ~' 5 6 pGT/US95/148 Table 5.
PROBE 518 582 RQ- RQ+ 0 RQ
no +temp. no temp. +temp. -temp.
Al-PS 149 354 253 379 0.42 0.67 0.25 - -Al-CPG 494 437 1500 616 1.13 2.44 1.31 Gl-PS 75 166 178 245 0.45 0.73 0.28 5. Method For Monitoring PCR Amplification sing Oli;~~nucleotide Probe All PCR amplifications were performed in a Perkin-Elmer Thermocycler 9600 using 50 p.l reactions that contained 10 mM°Tris-HCl (pH 8.3), 50 mM
KCI, 200 ~.M
dATP, 200 p,M dCTP, 200 ~.M dGTP, 400 ~,M dUTP, 0.5 units AmpEraseTM uracil N-glycolyase (Perkin-Elmer), and 1.25 units AmpliTaqTM (Perkin-Elmer). A 295 basepair segment of exon 3 of human ~-actin gene (nucleotides 2141-2435 disclosed by Nakajima-Iijima, S., P_roc. Natl. Acad Sci SA 82: 6133-6137 (1985) was amplified using the AFP and ARP primers listed below. The amplification reactions contained 4 mM MgCl2, 20 ng human genomic DNA, 50 nM A1 or A3 probe, and 300 nM of each primer. Thermal regimen was 50 °C (2 min); 95 °C (10 min); 40 cycles of 95 °C (20 sec); 60 °C (1 min); and hold at 72 °C. A 515 basepair segment was amplified from a plasmid that consists of a segment of .1 DNA (nucleotides 32, 220-32, 747) inserted into the Sma I site of vector pUCl 19. These reactions contained 3.5 mM MgCI,, 1 ng plasmid DNA, 50 nMP2 or PS probe, 200 nM primer F 119, and 200 nM primer 8119.
The thermal regimen was 50 °C (2 min); 95 °C (10 min); 25 cycles of 95 °C (20 sec), 57 °C (1 min); and hold at 72 °C.
For each amplification reaction, 40 ~.l was transferred to an individual well of a white 96-well microtiter plate (Perkin-Elmer). Fluorescence was measured on a Perkin-Elmer TaqManTM LS-SOB System, which consists of a luminescence spectrometer with a plate reader assembly, a 485 nm excitation filter, and a 515 nm emission filter.
WO 96/15270 PCTlUS95/14882 Excitation was carried out at 488 nm using a 5 nm slit width. Emission was measured at 518 nm for 6-FAM (the reporter, or R Valve) and 582 nm for TAMR.A (the quencher, or Q value) using a 10 nm slit width. In order to determine the increase in reporter emission ' that is due to cleavage of the probe during PCR, three normalizations are applied to the raw emission data. First, emission intensity of a buffer blank is subtracted for each wavelength. Second, emission intensity of the reporter is divided by the emission intensity of the quencher to give an RQ ratio for each reaction tube. This normalizes for well-to-well variation in probe concentration and fluorescence measurement.
Finally, ~RQ is calculated by subtracting the RQ value of the no template control (RQ') from the RQ value for the complete reaction including a template (RQ~.
Three pairs of probes were tested in PCR assays. For each pair, one probe has TAMRA attached to an internal nucleotide and the other has TAMRA attached to the 3' end nucleotide. Results are shown in Table 6. For all three sets, the probe with the 3' quencher exhibits a ORQ value that is considerable higher than for the probe with the internal quencher.
Table 6.
PROBE 518 582 RQ- RQ+ ORQ
no temp. +temp. no temp. +temp.
A3-6 34.06 50.1 73.78 70.8 0.5 0.71 0.25 A3-24 58.85 202 69.66 78.8 0.8 2.57 1.72 P2-7 67.58 341 85.78 87.9 0.8 3.89 3.1 P2-27 124.6 722 152.6 118 0.8 6.1 5.28 PS-10 77.32 156 75.41 67 1 2.33 1.3 PS-28 73.23 507 106.6 96.3 0.7 5.28 4.59 WO 96/15270 ~ 7 PGT/US95/14882 Table 7. Fluorescence In Single And Double-stranded States.
Probe 518 582 RQ
ss ds ss ds ss ds - --PZ-7 63.81 84.07 96.52 142.97 0.66 0.59 _ _ P2-27 92.31 557.53 165.13 89.47 0.56 6.23 PS-10 266.30 366.37 437.97 491.00 0.61 0.75 PS-28 51.91 782.80 141.20 154.07 0.37 5.08 Al-7 18.40 60.45 105.53 218.83 0.17 0.28 Al-26 87.75 734.37 90.91 118.57 0.97 6.19 44.77 104.80 90.80 177.87 0.49 0.59 A3-24 45.57 857.57 100.15 191.43 0.46 3.47 Table 7 gives the results of fluorescence measurements of the indicated probes in single and double-stranded states. For probes having reporter and quencher at opposite ends of the oligonucleotide, hybridization caused a dramatic increase in RQ.
The foregoing description of a preferred embodiment of the invention has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise forms disclosed. Obviously, many modifications and variations will be apparent to practitioners skilled in this art. It is intended that the scope of the invention be defined by the following claims and their equivalents.
WO 96/15270 ~ ~ ~ PCT/US95/14882 SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Perkin-Elmer Corporation, Applied Biosystems Division (ii) TITLE OF INVENTION: SELF-QUENCHING FLUORESCENCE
PROBE AND METHOD
(iii) NUMBER OF SEQUENCES: 14 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: David J. Weitz, Haynes & Davis (B) STREET: 2180 Sand Hill Road, Suite 310 (C) CITY: Menlo Park (D) STATE: California (E) COUNTRY: USA
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(A) NAME: David J. Weitz (B) REGISTRATION NUMBER: 38,362 (C) REFERENCE/DOCKET NUMBER: PELM4264CIP1W0 (ix) TELECOMMUNICATION INFORMATION:
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
2201~~~
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
(2) INFORMATION FOR SEQ ID NO: 4 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4 (2) INFORMATION FOR SEQ ID NO: 5 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5 CGGATTTGCT GGTATCTATG ACAAGGAT 2g (2) INFORMATION FOR SEQ ID NO: 6 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6 _ (2) INFORMATION FOR SEQ ID NO: 7 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7 (2) INFORMATION FOR SEQ ID NO: 8 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8 (2) INFORMATION FOR SEQ ID NO: 9 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single zzo~7~~
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9 (2) INFORMATION FOR SEQ ID NO: 10 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single ( D ) TOPOLOGY : 1 inear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10 (2) INFORMATION FOR SEQ ID NO: 11 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11 (2) INFORMATION FOR SEQ ID NO: 12 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12 WO 96!15270 (2) INFORMATION FOR SEQ ID NO: 13 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13 (2) INFORMATION FOR SEQ ID NO: 14 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14
That is, RQ' is the ratio of the fluorescent emissions of the reporter molecule to the fluorescence of the quencher molecule when the oligonucleotide probe is in a single-stranded state. Influences on the value of RQ' include, for example, the particular reporter and quencher molecules used, the spacing between the reporter and quencher molecules, nucleotide sequence-specific effects, and the degree of flexibility of structures, e.g., linkers, to which the reporter and quencher molecules are attached, and the presence of impurities. Wo et al., Anal. Biochem., 218: 1-13 (1994); and Clegg, Meth. Enzymol., 21 l: 353-388 (1992). A related quantity RQ+, refers to the ratio of fluorescent emissions of the reporter molecule to the quencher molecule when the oligonucleotide probe is hybridized to a complementary polynucleotide.
A second factor is the efficiency of the probe to hybridize to a complementary polynucleotide. This second factor depends on the probe's melting temperature, Tm, the presence of a secondary structure in the probe or target polynucleotide, the annealing temperature, and other reaction conditions.
A third factor is the efficiency with which the DNA polymerase 5'-~3' exonuclease activity cleaves the bound probe between the reporter molecule and quencher molecule. This efficiency depends on such factors as the proximity of the reporter or quencher to the 5' end of the probe, the "bulkiness" of the reporter or quencher, and the degree of complementarity between the probe and target polynucleotide. Lee et al., Nucleic Acids Research, 21: 3761-3766 ( 1993).
Since quenching depends on the physical proximity of the reporter molecule to the quencher molecule, it was previously assumed that the quencher ~~a~7~~
WO 96/15270 PGT/US95/1488J~
and reporter molecules must be attached to the probe such that the quencher molecule remains at all times within the maximum distance at which the quencher molecule can quench the reporter molecule, this distance generally being a separation of about 6-16 nucleotides. Lee et al. Nucleic Acids ' S Research, 21: 3761-3766 (1993); Mergny et al., Nucleic Acids Research 22:
920-928 (1994); Cardullo et al., Proc. Natl. Acad. Sci., 85: 8790-8794 (1988);
Clegg et al., Proc. Natl. Acad. Sci., 90: 2994-2998 (1993); and Ozaki et al., Nucleic Acids Research, 20: 5205-5214 (1992). This short separation between the reporter molecule and the quencher molecule is typically achieved by attaching one member of the reporter-quencher pair to the 3' or 5' end of the probe and the other member to an internal base 6-16 nucleotides away.
There are at least two significant disadvantages associated with attaching a reporter or quencher molecule to an internal base. Attaching a reporter or quencher molecule to an internal nucleotide typically involves more difficult chemistry than the chemistry required to attach the molecule to a terminal nucleotide. In addition, attachment of a reporter or quencher molecule to an internal nucleotide can adversely affect the hybridization efficiency of the probe. Ward et al., U. S. Patent 5,328,824; and Ozaki et al. Nucleic Acids Research, 20: 5205-5214 (1992).
A need currently exists for effective oligonucleotide probes containing a fluorescent reporter molecule and a quencher molecule for use in hybridization and nucleic acid amplification assays. Accordingly, a need exists for probes which exhibit distinguishable fluorescence characteristics when hybridized and not hybridized to a target nucleic acid sequence. A further need exists for probes where the reporter molecule and quencher molecule are positioned on the probe such that the quencher molecule can effectively quench the fluorescence of the reporter molecule. A further need exists for probes which are efficiently synthesized. Yet a further need exists for the reporter molecule and quencher molecule to be positionable on the probe such that the reporter and quencher molecules do not adversely impact the hybridization efficiency of probe. These 2~017~6 and further objectives are provided by the probes and methods of the present invention.
~IVIMARY OF THE INVENTION
The present invention relates to an oli~onucle~tirie "rr,hP .x~h;..h ;.".~.,ao~
o------------- t..a...... ..aaavaa laavluucJ
a fluorescent reporter molecule and a quencher molecule capable of quenching the fluorescence of the reporter molecule. According to the present invention, the oligonucleotide probe is constructed such that the probe exists in at least one single-stranded conformation when unhybridized where the quencher molecule is near enough to the reporter molecule to quench the fluorescence of the reporter molecule. The oligonucleotide probe also exists in at least one conformation when hybridized to a target polynucleotide where the quencher molecule is not positioned close enough to the reporter molecule to quench the fluorescence of the reporter molecule. By adopting these hybridized and unhybridized conformations, the reporter molecule and quencher molecule on the probe exhibit different fluorescence signal intensities when the probe is hybridized and unhybridized. As a result, it is possible to determine whether the probe is hybridized or unhybridized based on a change in the fluorescence intensity of the reporter molecule, the quencher molecule, or a combination thereof. In addition, because the probe can be designed such that the quencher molecule quenches the reporter molecule when the probe is not hybridized, the probe can be designed such that the reporter molecule exhibits limited fluorescence until the probe is either hybridized or digested.
According to the present invention, the fluorescence intensity of the reporter molecule is preferably greater than the fluorescence intensity of the quencher molecule when the probe is hybridized to the target polynucleotide.
The fluorescence intensity of the reporter molecule is more preferably at least about a factor of 3.5 greater than the fluorescence intensity of the quencher molecule when the probe is hybridized to the target polynucleotide.
The fluorescence intensity of the oligonucleotide probe hybridized to the target polynucleotide is also preferably at least about a factor of 6 greater than the fluorescence intensity of the oligonucleotide probe when not hybridized to the target polynucleotide.
The reporter molecule is preferably separated from the quencher _ molecule by at least about 15 nucleotides, more preferably at least about 18 nucleotides. The reporter molecule is preferably separated from the quencher molecule by between about 15 and 60 nucleotides, more preferably between about 18 and 30 nucleotides.
The reporter molecule is preferably attached to either the 3' or 5' terminal nucleotides of the probe. The quencher molecule is also preferably attached to either the 3' or 5' terminal nucleotides of the probe. More preferably, the reporter and quencher molecules are attached to the 3' and 5' or 5' and 3' terminal nucleotides of the probe respectively.
The reporter molecule is preferably a fluorescein dye and the quencher molecule is preferably a rhodamine dye.
In one embodiment, the oligonucleotide probe of the present invention is immobilized on a solid support. The oligonucleotide probe may be attached directly to the solid support, for example by attachment of the 3' or 5' terminal nucleotide of the probe to the solid support. More preferably, however, the probe is attached to the solid support by a linker. The linker serves to distance the probe from the solid support. The linker is most preferably at least 30 atoms in length, more preferably at least 50 atoms in length.
A wide variety of linkers are known in the art which may be used to attach the oligonucleotide probe to the solid support. The linker most preferably includes a functionalized polyethylene glycol because it does not significantly interfere with the hybridization of probe to the target oligonucleotide, is commercially available, soluble in both organic and aqueous media, easy to functionalize, and completely stable under oligonucleotide synthesis and post-synthesis conditions.
_8_ The linkages between the solid support, the linker and the probe are preferably not cleaved during removal of base protecting groups under basic conditions at high temperature. Examples of preferred linkages include carbamate and amide linkages.
The present invention also relates to the use of the oligonucleotide probe _ _ as a hybridization probe to detect target polynucleotides. Accordingly, the present invention relates to a hybridization assay for detecting the presence of a target polynucleotide in a sample. In one embodiment of the method, the hybridization probe is immobilized on a solid support.
According to the method, an oligonucleotide probe of the present invention is contacted with a sample of polynucleotides under conditions favorable for hybridization. The fluorescence signal of the reporter molecule before and after being contacted with the sample is compared. Since the reporter molecule on the probe exhibits a greater fluorescence signal when hybridized to a target sequence, an increase in the fluorescence signal after the probe is contacted with the sample indicates the hybridization of the probe to target sequences in the sample, thereby indicating the pressure of target sequences in the sample. Quantification of the change in fluorescence intensity as a result of the probe being contacted with the sample can be used to quantify the amount of target sequences present in the sample.
The present invention also relates to the use of the oligonucleotide probe for monitoring nucleic acid amplification. Accordingly, the present invention relates to a method for monitoring nucleic acid amplification by performing nucleic acid amplification on a target sequence using a nucleic acid polymerase having 5', 3' nuclease activity, a primer capable of hybridizing to the target sequence and an oligonucleotide probe according to the present invention which is capable of hybridizing to the target sequence 3' relative to the primer.
According to the method, the nucleic acid polymerase digests the oligonucleotide probe during amplification when it is hybridized to the target sequence, thereby separating the reporter molecule from the quencher molecule.
As the amplification is conducted, the fluorescence of the reporter molecule is monitored, the generation of fluorescence corresponding to the occurrence of nucleic acid amplification. Accordingly, the amount of amplification performed can be quantified based on the change in fluorescence observed. It is noted that the fluorescence of the quencher molecule may also be monitored, either separately or in combination with the reporter molecule to detect amplification.
According to an aspect of the invention, there is provided a method of nucleic acid amplification comprising:
performing nucleic acid amplification on a target polynucleotide using a nucleic acid polymerase having 5' to 3' nuclease activity, a primer capable of hybridizing to the target polynucleotide, and an oligonucleotide probe under amplification conditions such that the probe hybridizes to the target polynucleotide 3' relative to the primer and the probe does not hybridize with itself to form a hairpin structure, the oligonucleotide probe having at one end a fluorescent reporter and at the other end a quencher that quenches the fluorescence of the reporter molecule when both the fluorescent reporter and quencher are attached to the probe, under conditions such that digestion of the oligonucleotide probe by the polymerase during amplification is effective to separate the reporter from the quencher, whereby a fluorescence signal of the reporter is increased.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 illustrates a method for real-time monitoring nucleic acid amplification utilizing a probe which is degraded by the 5'~3' exonuclease activity of a nucleic acid polymerase.
Figure 2 illustrates a probe according to the present invention immobilized to a solid support in hybridized and unhybridized conformations.
- l0a -DETAILED DESCRIPTION
The present invention relates to an oligonucleotide probe which includes a fluorescent reporter molecule and a quencher molecule capable of quenching the fluorescence of the reporter molecule. According to the present invention, the oligonucleotide probe is constructed such that the probe exists in at least one single-stranded conformation when unhybridized where the quencher molecule is near enough to the reporter molecule to quench the fluorescence of the reporter molecule.
The oligonucleotide probe also exists in at least one conformation when hybridized to a target polynucleotide such that the quencher molecule is not positioned close enough to the reporter molecule to quench the fluorescence of the reporter molecule.
By adopting these hybridized and unhybridized conformations, the reporter molecule and quencher molecule on the probe exhibit different fluorescent signal intensities when the probe is hybridized and unhybridized. As a result, it is possible to determine whether the probe is hybridized or unhybridized based on a change in the fluorescence intensity of the reporter molecule, the quencher molecule, or a combination thereof. In addition, because the probe can be designed such that the quencher molecule quenches the reporter molecule when the probe is not hybridized, the probe can be designed such that the reporter molecule exhibits limited fluorescence unless the probe is either hybridized or digested.
According to the present invention, the fluorescence intensity of the reporter molecule is preferably greater than the fluorescence intensity of the quencher molecule when the probe is hybridized to the target polynucleotide.
The fluorescence intensity of the reporter molecule is more preferably at least about a factor of 3.5 greater than the fluorescence intensity of the quencher molecule when the probe is hybridized to the target polynucleotide.
The fluorescence intensity of the oligonucleotide probe hybridized to the target polynucleotide is also preferably at least about a factor of 6 greater than the fluorescence intensity of the oligonucleotide probe when not hybridized to the target polynucleotide.
The reporter molecule is preferably separated from the quencher molecule by at least about 15 nucleotides, more preferably at least about 18 nucleotides. The reporter molecule is preferably separated from the quencher molecule by between about 15 and 60 nucleotides, more preferably between about 18 and 30 nucleotides.
The reporter molecule is preferably attached to either the 3' or 5' terminal nucleotides of the probe. The quencher molecule is also preferably attached to either the 3' or 5' terminal nucleotides of the probe. More preferably, the reporter and quencher molecules are attached to the 3' and 5' or 5' and 3' terminal nucleotides of the probe respectively.
The reporter molecule is preferably a fluorescein dye and the quencher molecule is preferably a rhodamine dye.
In one embodiment, the oligonucleotide probe is attached to a solid support. As illustrated in Figure 2, when the probe is unhybridized, the probe is able to adopt at least one single-stranded conformation such that the quencher molecule is near enough to the reporter molecule to quench the fluorescence of WO 96/15270 2 ~ Q 17 5 6 pCT/US95114882~
the reporter molecule. As further illustrated in Figure 2, when the probe is hybridized to a target sequence, the probe adopts at least one conformation where the quencher molecule is not positioned close enough to the reporter , molecule to quench the fluorescence of the reporter molecule. As a result, the fluorescence intensity of the reporter molecule increases when the probe . ' hybridizes to a target sequence.
As illustrated in Figure 2, different probes may be attached to the solid support and may be used to simultaneously detect different target sequences in a sample. Reporter molecules having different fluorescence wavelengths can be used on the different probes, thus enabling hybridization to the different probes to be separately detected.
Examples of preferred types of solid supports for immobilization of the oligonucleotide probe include controlled pore glass, glass plates, polystyrene, avidin coated polystyrene beads, cellulose, nylon, acrylamide gel and activated dextran. CPG, glass plates and high cross-linked polystyrene. These solid supports are preferred for hybridization and diagnostic studies because of their chemical stability, ease of functionalization and well defined surface area.
Solid supports such as controlled pore glass (CPG, 500 A, 1000 ~) and non-swelling high cross-linked polystyrene (1000 t~) are particularly preferred in view of their compatibility with oligonucleotide synthesis.
The oligonucleotide probe may be attached to the solid support in a variety of manners. For example, the probe may be attached to the solid support by attachment of the 3' or 5' terminal nucleotide of the probe to the solid support. More preferably, however, the probe is attached to the solid support by a linker which serves to distance the probe from the solid support. The linker is most preferably at least 30 atoms in length, more preferably at least 50 atoms in length.
The length and chemical stability of linker between solid support and the first 3' unit of oligonucleotides play an important role in efficient synthesis and hybridization of support bound oligonucleotides. The linker arm should be zzo~~~s sufficiently long so that a high yield (>97%) can be achieved during automated synthesis. The required length of the linker will depend on the particular solid support used. For example, a six atom linker is generally sufficient to achieve a >97% yield during automated synthesis of oligonucleotides when high cross-linked polystyrene is used as the solid support. The linker arm is preferably at least 20 atoms long in order to attain a high yield (>97%) during automated synthesis when CPG is used as the solid support.
Hybridization of a probe immobilized to a solid support generally requires that the probe be separated from the solid support by at least 30 atoms, more preferably at least 50 atoms. In order to achieve this separation, the linker generally includes a spacer positioned between the linker and the 3' nucleoside.
For oligonucleotide synthesis, the linker arm is usually attached to the 3'-OH
of the 3' nucleoside by an ester linkage which can be cleaved with basic reagents to free the oligonucleotide from the solid support.
A wide variety of linkers are known in the art which may be used to attach the oligonucleotide probe to the solid support. The linker may be formed of any compound which does not significantly interfere with the hybridization of the target sequence to the probe attached to the solid support. The linker may be formed of a homopolymeric oligonucleotide which can be readily added on to the linker by automated synthesis. Alternatively, polymers such as functionalized polyethylene glycol can be used as the linker. Such polymers are preferred over homopolymeric oligonucleotides because they do not significantly interfere with the hybridization of probe to the target oligonucleotide. Polyethylene glycol is particularly preferred because it is commercially available, soluble in both organic and aqueous media, easy to functionalize, and completely stable under oligonucleotide synthesis and post-synthesis conditions.
The linkages between the solid support, the linker and the probe are preferably not cleaved during removal of base protecting groups under basic WO 96/15270 ~ 2 Q 1 ~ 5 6 PCT/US951i488~
conditions at high temperature. Examples of preferred linkages include carbamate and amide linkages.
The oligonucleotide probe of the present invention may be used as a hybridization probe to detect target polynucleotides. Accordingly, the present invention relates to a hybridization assay for detecting the presence of a target _ ' polynucleotide in a sample. According to the method, an oligonucleotide probe of the present invention is contacted with a sample of nucleic acids under conditions favorable for hybridization. The fluorescence signal of the reporter molecule is measured before and after being contacted with the sample. Since the reporter molecule on the probe exhibits a greater fluorescence signal when hybridized to a target sequence, an increase in the fluorescence signal after the probe is contacted with the sample indicates the hybridization of the probe to target sequences in the sample and hence the presence of target sequences in the sample. Further, by quantifying the change in fluorescence intensity as a result of the probe being contacted with the sample, the amount of target sequences in the sample can be quantified.
According to one embodiment of the method, the hybridization probe is immobilized on a solid support. The oligonucleotide probe is contacted with a sample of nucleic acids under conditions favorable for hybridization. The fluorescence signal of the reporter molecule is measured before and after being contacted with the sample. Since the reporter molecule on the probe exhibits a greater fluorescence signal when hybridized to a target sequence, an increase in the fluorescence signal after the probe is contacted with the sample indicates the hybridization of the probe to target sequences in the sample. Immobilization of the hybridization probe to the solid support enables the target sequence hybridized to the probe to be readily isolated from the sample. In later steps, the isolated target sequence may be separated from the solid support and processed ' (e.g., purified, amplified) according to methods well known in the art depending on the particular needs of the researcher.
The oligonucleotide probe of the present invention may also be used as a probe for monitoring nucleic acid amplification. Accordingly, the present invention relates to a method for monitoring nucleic acid-amplification using an oligonucleotide probe according to the present invention which is capable of hybridizing to the target sequence 3' relative to an ampIif cation primer.
According to the method, nucleic acid amplification is performed on a target polynucleotide using a nucleic acid polymerise having 5'- 3' nuclease activity, a primer capable of hybridizing to the target polynucleotide, and an oligonucleotide probe according to the present invention capable of hybridizing to the target polynucleotide 3' relative to the primer. During amplification, the nucleic acid polymerise digests the oligonucleotide probe when it is hybridized to the target sequence, thereby separating the reporter molecule from the quencher molecule. As the amplification is conducted, the fluorescence of the reporter molecule is monitored, the generation of fluorescence corresponding to 1 S the occurrence of nucleic acid amplification.
Use of a reporter-quencher pair probe generally in conjunction with the amplification of a target polynucleotide, for example, by PCR, e.g., is described in many references, such as Innis et al., editors, PCR Protocols (Academic Press, New York, 1989); Sambrook et al., Molecular Cloning, Second Edition (Cold Spring Harbor Laboratory, New York, 1989).
The binding site of the oligonucleotide probe is located between the PCR primers used to amplify the target polynucleotide.
Preferably, PCR is carried out using Taq DNA polymerise, e.g., AmplitaqTM
(Perkin-Elmer, Norwalk, CN), or an equivalent thermostable DNA polymerise, and the annealing temperature of the PCR is about ~-10°C below the melting temperature of the oligonucleotide probes employed.
Use of an oligonucleotide probe according to the present invention for monitoring nucleic acid amplification provides several advantages over the use of prior art reporter-quencher pair probes. For e:cample, prior art probes required that the reporter and quencher molecules be positioned on the probe - I~-such that the quencher molecule remained within a minimum quenching distance of the reporter molecule. However, by realizing that the probe need only be designed such that the probe be able to adopt a conformation where the quencher molecule is within a minimum quenching distance of the reporter molecule, a far wider array of probes are enabled. For example, dually labelled probes having the reporter and quencher molecules at the 5' and 3' ends can be designed. Such probes are far easier to synthesize than probes where the reporter molecule or the quencher molecule is attached to an internal nucleotide.
Positioning of the reporter and quencher molecules on terminal nucleotides also enhances the hybridization efficiency of the probes.
As used in this application, the term "oligonucleotide", includes linear oligomers of natural or modified monomers or linkages, including deoxyribonucleosides, ribonucleosides, and the like; capable of specifically binding a target polynucleotide by way of a regular pattern of monomer-to-monomer interactions, such as Watson-Crick type of basepairing, or the like.
Usually monomers are linked by phosphodiester bonds or analogs thereof to form oligonucleotides ranging in size from a few monomeric units, e.g., 3-4, to several tens of monomeric units. Whenever an oligonucleotide is represented by a sequence of letters, such as "ATGCCTG", it will be understood that the nucleotides are in 5' --~ 3' order from left to right and that "A" denotes deoxyadenosine, "C" denotes deoxycytidine, "G" denotes deoxyguanosine, and "T" denotes thymidine, unless otherwise noted. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoranilidate, phosphoramidate, and the like. Generally, oligonucleotide probes of the invention will have a sufficient number of phosphodiester linkages adjacent to its 5' end so that the 5' -~ 3' exonuclease activity employed can efficiently degrade the bound probe to separate the reporter and quencher molecules.
"Perfectly matched" in reference to a duplex means that the poly- or oligonucleotide strands making up the duplex form a double-stranded structure with one other such that every nucleotide in each strand undergoes Watson-22Q175~
Crick basepairing with a nucleotide in the other strand. The term also comprehends the pairing of nucleoside analogs, such as deoxyinosine, nucleosides with 2-aminopurine bases, and the like, that may be employed.
Conversely, a "mismatch" in a duplex between a target polynucleotide and an oligonucleotide probe or primer means that a pair of nucleotides in the duplex fails to undergo Watson-Crick bonding.
As used in the application, "nucleoside" includes the natural nucleosides, including 2'-deoxy and 2'-hydroxyl forms, e.g., as described in Kornberg and Baker, DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992). "Analogs" in reference to nucleosides includes synthetic nucleosides having modified base moieties and/or modified sugar moieties, e.g., described by Scheit, Nucleotide Analogs (John Wiley, New York, 1980); Uhlman and Peyman, Chemical Reviews, 90: 543-584 (1990), or the like, with the only proviso that they are capable of specific hybridization. Such analogs include synthetic nucleosides designed to enhance binding properties, reduce degeneracy, increase specificity, and the like.
Oligonucleotide probes of the invention can be synthesized by a number of approaches, e.g., Ozaki et al., Nucleic Acids Research, 20: 5205-5214 (1992); Agrawal et al., Nucleic Acids Research, 18: 5419-5423 (1990); or the like. The oligonucleotide probes of the invention are conveniently synthesized on an automated DNA synthesizer, e.g., an Applied Biosystems, Inc. (Foster City, California) model 392 or 394 DNA/RNA Synthesizer, using standard chemistries, such as phosphoramidite chemistry, e.g., disclosed in the following references: Beaucage and Iyer, Tetrahedron, 48: 2223-2311 (1992); Molko et al., U. S. Patent 4,980,460; Koster et al., U. S. Patent 4,725,677; Caruthers et al., U. S. Patents 4,415,732; 4,458,066; and 4,973,679; and the like.
Alternative chemistries, e.g., resulting in non-natural backbone groups, such as phosphorothioate, phosphoramidate, and the like, may also be employed provided that the hybridization efficiencies of the resulting oligonucleotides WO 96/15270 PCT/US95/14882~
and/or cleavage efficiency of the exonuclease employed are not adversely affected.
Preferably, the oligonucleotide probe is in the range of 15-60 nucleotides in length. More preferably, the oligonucleotide probe is in the range of 18-30 nucleotides in length. The precise sequence and length of an oligonucleotide probe of the invention depends in part on the nature of the target polynucleotide to which it binds. The binding location and length may be varied to achieve appropriate annealing and melting properties for a particular embodiment.
Guidance for making such design choices can be found in many of the above-cited references describing the "Taq-man" type of assays.
Preferably, the 3' terminal nucleotide of the oligonucleotide probe is blocked or rendered incapable of extension by a nucleic acid polymerase. Such blocking is conveniently carried out by the attachment of a reporter or quencher molecule to the terminal 3' carbon of the oligonucleotide probe by a linking moiety.
Preferably, reporter molecules are fluorescent organic dyes derivatized for attachment to the terminal 3' carbon or terminal 5' carbon of the probe via a linking moiety. Preferably, quencher molecules are also organic dyes, which may or may not be fluorescent, depending on the embodiment of the invention.
~ For example, in a preferred embodiment of the invention, the quencher molecule is fluorescent. Generally whether the quencher molecule is fluorescent or simply releases the transferred energy from the reporter by non-radiative decay, the absorption band of the quencher should substantially overlap the fluorescent emission band of the reporter molecule. Non-fluorescent quencher molecules that absorb energy from excited reporter molecules, but which do not release the energy radiatively, are referred to in the application as chromogenic molecules.
There is a great deal of practical guidance available in the literature for selecting appropriate reporter-quencher pairs for particular probes, as exemplified by the following references: Clegg (cited above); Wu et al. (cited above); Pesce et al., editors, Fluorescence Spectroscopy (Marcel Dekker, New York, 1971); White et al., Fluorescence Analysis: A Practical Approach (Marcel Dekker, New York, 1970); and the like. The literature also includes references providing exhaustive lists of fluorescent and chromogenic molecules and their relevant optical properties for choosing reporter-quencher pairs, e.g., Berlman, Handbook of Fluorescence Spectra of Aromatic Molecules, 2nd Edition (Academic Press, New York, 1971); Griffiths, Colour and Constitution of Organic Molecules (Academic Press, New York, 1976); Bishop, editor, Indicators (Pergamon Press, Oxford, 1972); Haugland, Handbook of Fluorescent Probes and Research Chemicals (Molecular Probes, Eugene, 1992) Pringsheim, Fluorescence and Phosphorescence (Interscience Publishers, New York, 1949); and the like. Further, there is extensive guidance in the literature for derivatizing reporter and quencher molecules for covalent attachment via common reactive groups that can be added to an oligonucleotide, as exemplified by the following references: Haugland (cited above); Ullman et al., U. S.
Patent 3,996,345; Khanna et al., U. S. Patent 4,351,760; and the like.
Exemplary reporter-quencher pairs may be selected from xanthene dyes, including fluoresceins, and rhodamine dyes. Many suitable forms of these compounds are widely available commercially with substituents on their phenyl moieties which can be used as the site for bonding or as the bonding functionality for attachment to an oligonucleotide. Another group of fluorescent compounds are the naphthylamines, having an amino group in the alpha or beta position. Included among such naphthylamino compounds are 1-dimethylaminonaphthyl-S-sulfonate, 1-anilino-8-naphthalene sulfonate and 2-p-touidinyl-6-naphthalene sulfonate. Other dyes include 3-phenyl-7-isocyanatocoumarin, acridines, such as 9-isothiocyanatoacridine and acridine orange; N-(p-(2-benzoxazolyl)phenyl)maleimide; benzoxadiazoles, stilbenes, pyrenes, and the like.
Preferably, reporter and quencher molecules are selected from fluorescein and rhodamine dyes. These dyes and appropriate linking methodologies for attachment to oligonucleotides are described in many references, e.g., Khanna et al. (cited above); Marshall, Histochemical J., 7:
303 (1975); Menchen et al., U. S. Patent 5,188,934; Mencl~en et al., European Patent Application 87310256.0; and Bergot et al., International Application PCT/US90/05565.
There are many linking moieties and methodologies for attaching reporter or quencher molecules to the 5' or 3' termini of oIigonucleotides, as exemplified by the following references: Eckstein, editor, Oligonucleotides and Analogues: A Practical Approach (IRL Press, Oxford, 1991); Zuckerman et al., Nucleic Acids Research, 15: 5305-5321 (1987) (3' thiol group on oligonucleotide); Sharma et al., Nucleic Acids Research, 19: 3019 (1991) (3' sulflnydryl); Giusti et al., PCR Methods and Applications, 2: 223-227 (1993) and Fung et al., U. S. Patent 4, 757,141 (5' phosphoamino group via AminolinkTM II available from Applied Biosystems, Foster City, CA) Stabinsky, U. S. Patent 4,739,044 (3' aminoalkylphosphoryl group); Agrawal et al., Tetrahedron Letters, 31: 1543-1546 (1990) (attachment via phosphoramidate linkages); Sproat et al., Nucleic Acids Research, 15: 4837 (1987) (5' mercapto group); Nelson et al., Nucleic Acids Research, 17: 7187-7194 ( 1989) (3' amino group); and the like.
Preferably, commercially available linking moieties are employed that can be attached to an oligonucleotide during synthesis; e.g., available from Clontech Laboratories (Palo Alto, CA).
Rhodamine and fluorescein dyes are also conveniently attached to the 5' hydroxyl of an oIigonucleotide at the conclusion of solid phase synthesis by way of dyes derivatized with a phosphoramidite moiety, e.g., Woo et al., U. S.
Patent 5,231, 191; and Hobbs, Jr., U. S. Patent 4,997,928.
The following examples set forth probes and methods for using the probes according to the present invention. It is understood that the specific probes, probe constructs and steps of the methods described in these examples are,not intended to be limiting. Further objectives and advantages of the present invention other than those set forth above will become apparent from the examples which are not intended to limit the scope of the present invention.
EXAMPLES
1. SmtheSis n Oli~onucleotide Probes The following example describes the synthesis of the oligonucleotides shown in Table 1. Linker arm nucleotide ("LAN") phosphoramidite was obtained from Glen Research. Standard DNA phosphoramidites, 6-carboxyfluorescein ("6-FAM") phosphoramidite, 6- ...
carboxytetramethyIrhodamine succinimidyl ester ("TAMRA NHS ester"), and PhosphalinkTM for attaching a 3' blocking phosphate were obtained from Perkin-Elmer, Applied Biosystems Division. Oligonucleotide synthesis was performed on a model 394 DNA Synthesizer (Applied Biosystems). Primer and complement oIigonucleotides were purified using OIigo Purification Cartridges (Applied Biosystems).. Doubly labeled probes were synthesized with 6-FAM-labeled phosphoramidite at the 5' end, LAN replacing one of the T's in the oligonucIeotide sequence, and PhosphalinkTM at the 3' end. Following deprotection and ethanol precipitation, TAMRA NHS ester was coupled to the LAN-containing oligonucleotide in 250 mM Na-bicarbonate buffer (pH 9.0) at room temperature. Unreacted dye was removed by passage over a PD-10 Sephadex column. Finally, the doubly labeled probe was purified by preparative HPLC using standard protocols. Below, probes are named by designating the sequence from Table I and the position of the LAN-TAMRA
moiety. For example, probe AI-7 has sequence of AI with LAN-TAMR.A at nucleoside position 7 from the 5' end.
*Trademark WO 96/15270 ~ 2 Q 1 7 5 ~ pGT/US95/14882 Table 1. Sequences of oligonucleotides Name Type Sequence F 119 primer ACCCACAGGAACTGATCACCACTC
[SEQ. ID. No.: 1 ]
8119 primer ATGTCGCGTTCCGGCTGACGTTCTGC
[SEQ. ID. No.: 2]
P2 probe TCGCAT~ACTGATCGTTGCCAACCAGTp [SEQ. ID. No.: 3]
P2C complement GTACTGGTTGGCAACGATCAGTAATGCGATG
[SEQ. ID. No.: 4] .
PS probe CGGATTTGCTGGTATCTATGACAAGGATp [SEQ. ID. No.: 5]
PSC complement TTCATCCTTGTCATAGATACCAGCAAATCCG
[SEQ. ID. No.: 6]
AFP primer TCACCCACACTGTGCCCATCTACGA
[SEQ. ID. No.: 7]
ARP primer CAGCGGAACCGCTCATTGCCAATGG
[SEQ. ID. No.: 8]
Al probe A_TGCCC_TCCCCCA_TGCCA_TCC_TGCGTp [SEQ. ID. No.: 9]
A1C complement AGACGCAGGATGGCATGGGGGAGGGCATAC
[SEQ. ID. No.: 10]
A3 probe CGCCCTGGACTTCGAGCAAGAGAT~
[SEQ. ID. No.: 11]
A3C complement CCATCTCTTGCTCGAAGTCCAGGGCGAC
[SEQ. ID. No.: 12]
Gl probe CAAGCTTCCCGTTCTCAGCCT
[SEQ. ID. No.: 13]
G1C complement ACCGTCAAGGCTGAGAACGGGAAGCTTGTC
[SEQ. ID. No.: 14]
22n1756 Table 2.
O
NHFmoc HO
HOBT, HBTU, DIPEA, DL-Homoserine O
H~ NHFmoc N
DMT-Cl, DMAP, pyr O
H~ NHFmoc N
Polyethylene glycol) bis(2-aminoethyl ether), HOBT, HBTU, DIPEA, DMF
o H~ NHFmoc N
H
DMTO N
~ PEG-NH2 Succinic anhydride, DMAP, E~N,CH2C12 O
H~ HFmoc N
N/H /H O
DMT ~ ~ PEG-N H
O
WO 96/15270 pCT/LTSg5114882 CG
x.-z ~ x.-z i.-O ~
O
N N
x x U U
_ x Z
U
U
o O- U
x I I
x-z- U-= x U
U Z U
a O- U
O~. U ~
a a Z x_ Z .Q x- Z ~ ~' V
m ~ Z a a c I x i GL..'~ v ~ ~ m ~ .;
m x z a =~ Z suesn $
I ~ _ O-U ~
= O-U
~ a 'e a V ~ V 'm a N E' ~ ~ ~
' O-. sa.~ O= D
y x~Z _ x-z N
x ~I
r V
~/
v 2~0 ~~5s WO 96/15270 PCTlUS95/14882 L °
N
U IN
V ~ U
O=U Z I O=U Z
I
Z-Z-U-U c Z-Z- i U ._°.
o= I g O-U
z-z ~ z-z I
m 0. m c I a o D
O= ~ O-U
U
h N
N N O
a°
O=U ~ O=U
N z ( o z z-z ~ r~ z-z o a a v~
E= V ~ = V m m ..z a O=U = C O=U N ~ O=U x a Q ea ~
o -.
U O U ,_ U
a a>
O=U O=U ~ O=U
Z-Z Z-Z =-2 = U = = U = = U
U ( U U ~ U U I U
O-tn-O O-~n-O O-VJ-O
O O O
:. ~. ;r :r ::
a~
.o :a L
WO 96/15270 ~ pGT/U895/1488~
2. Synthesis of Oli~onucleotide Probes Attached To A Solid Su_pnort Both high cross-linked polystyrene (1000 A) and controlled pore glass (CPG) (500 A) are used as solid support matrices. The functionalization of a spacer (compound S 5) is illustrated in Table 2. The attachment of the spacer to polystyrene and CPG
supports, and the labelling of the solid supports with TAMRA dye is shown in Tables 3 and 4 respectively.
Table 2 illustrates a reaction scheme for the synthesis of a spacer, compound 5, which is used to derivatize CPG and polystyrene supports. As shown in Table 2, N-Fmoc-e-aminocaproic acid was reacted with DL-homoserine in presence of HOBT/HBTU/DIPEA (Knorr, et al., Tetrahedron Lett. 1989, 30, 1927) in DMF to give compound 2 in 65% yield. Compound 2 was reacted with dimethoxytrityl chloride in presence of DMAP in pyridine to give compound 3 in 72% yield after chromatography.
Treatment of compound 3 with a large excess of PEG-diamine (Buckmann, et al., Biotech. Appl. Biochem 1987, 9, 258) in presence of HOBT/HBTU/DIPEA in DMF
afforded amine 4 in 60% yield. The amine 4 was then converted to succinate 5 by treating amine 4 with succinic anhydride/Et3N/DMAP in CHZCIz in 90% yield. The succinate 5 was then attached to polystyrene and CPG support as illustrated in Tables 3 and 4 respectively without further purification.
As illustrated in Tables 3 and 4, succinate 5 was separately reacted with polystyrene and CPG support in presence of HOBT/HBTU/DIPEA in DMF to provide functionalized support 6 (5 ~mol/g loading) and functionalized support 8 (15 ~mol/g loading) respectively. The Fmoc group was removed from support bound spacer by treating supports 6 and 8 with 20% piperidine in DMF (Fields, et al., JPeptide Res.
1990, 35, 161) to give amine which was reacted with TAMRA NHS ester to give TAMRA labeled supports 7 and 9 respectively. The polystyrene and CPG supports showed a final loading of 4.8 ~.mol/g and 14 ~,mol/g respectively by trityl cation assay.
Double labeled Taqman probe was synthesized using both TAMRA labeled supports 7 and 9, FastPhoramidites (User Bulletin Number 85, Perkin Elmer Corporation 1994) and FAM phosphoramidite (User Bulletin Number 78, Perkin Elmer Corporation zzo ~7~s 1994) in 40 nanomol scale. The support bound oligonucleotides were deprotected by treating with MeOHa-BuNH2:H20 (1:1:2) at 65 °C for 3 hours (Woo, et al., U.S. Patent No. 5,231,191). Liquid was removed and the support containing probes were washed with H20:MeOH (3:1) and MeOH. The support was then dried under vacuum and used in a hybridization assay. _ Experimental:
Compound 2: N,N Diisopropylethylamine (l.lg, 1.48 mL, 8.52 mmol), 1-hydroxybenzotriazol (420 mg, 3.1 mmol) and (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (1.17 g, 3.1 mmol) were added to a stirred solution of Nfmoc-e-aminocaproic acid (1 g, 2.84 mmol) in DMF (30 mL) at room temperature. After 15 min DL-homoserine (1.35 g, 11.36 mmol) was added to the reaction mixture. After 3 hours, DMF was removed under reduced pressure. The residue was dissolved in CHC13 (100 mL) and washed with 5% aqueous HCl (2 X 50 mL).
The organic layer was dried over MgS04 and evaporated to give a thick oil which was trituated with ether to give a colorless solid (840 mg, 65%). The product was left under high vacuum for 24 hours and used in the next step without fiufiher purification.
Compound 3: 4,4'-Dimethoxytrityl chloride (484mg, 1.43 mmol) and 4-dimethyaminopyridine (25mg, 0.2 mmol) were added to a stirred solution of compound 2 (500mg, 1.1 mmol) in pyridine (15 mL) at room temperature under nitrogen atmosphere.
After 14 hours, pyridine was removed and the residue was dissolved in CHCl3 (70 mL).
The organic layer was extracted with 5% aqueous citric acid (1 X 50 mL), H20 (1 X 50 mL) and saturated brine (1 X 50 mL). The organic layer was dried over MgS04 and evaporated to give a yellow foam. The product was purified by a silica gel column eluting with CHCl3-MeOH gradient (0-10% MeOH). The appropriate fractions were combined and evaporated to give Compound 3 as a colorless foam (600 mg, 72%).
_ Compound 4: Polyethylene glycol) bis(2-aminoethyl ether) (3.16 g, 5.3 mmol), N, N
diisopropylethylamine (205 mg, 0.27 mL, 1.59 mmol), 1-hydroxybenzotriazol (78 mg, wo 96ns2~o PCTlITS95/14882~
0.58 mmol) and (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluonium hexafluorophosphate (220 mg, 0.58 mmol) were added to a stirred solution of compound 3 (400 mg, 0.53 mmol) in DMF (10 mL) at room temperature. The reaction mixture was _ stirred at room temperature for 3 hours. DMF was removed under reduced pressure and, the residue was dissolved in CHCl3 (70 mL) and washed with H20 (1 X 50 mL) and saturated brine (2 X 50 mL). The organic layer was dried over MgS04 and evaporated to give a thick oil. Compound 4 was purified by a silica gel column eluting with a CHC13-MeOH gradient (5-15% MeOH) as a colorless glass (423 mg, 60%).
Compound 5: Succinic anhydride (22 mg, 0.22 mmol), Et3N (23 mg, 0.31 ~L, 0.22 mmol), 4-dimethylaminopyridine (14 mg, 0.11 mmol) were added to a solution of compound 4 (300 mg, 0.22 mmol) in CHzCIz (15 mL). The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was diluted with CHC13 (30 mL) and washed with 5% aqueous citric acid (1 X 50 mL) and saturated brine (2 X 50 mL).
The organic layer was dried over MgS04 and evaporated to a colorless foam (284 mg, 90%) which was used for derivatization of the solid support without further purification.
Derivatization of Polystyrene support with TAMRA dye: High cross linked polystyrene (1000 A, 10 ~,mol/g amine loading, lg, 10 ~mol), was treated with compound 5 (17 mg, 12 p,mol, .1-hydroxybenzotriazol (1.8 mg, 12 ~.mol), (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluonium hexafluorophosphate (4.8 mg, 12 ~mol), N,N diisopropylethylamine (6 ~L, 30 ~,mol) in DMF (10 mL) on a wrist action shaker for 4 hours at room temperature. The support was washed with DMF (3 X 10 mL), (2 X 10 mL) and CHZCIz (1 X 10 mL) and dried under high vacuum overnight. The ninhydrin assay showed 1 ~mol/g amine left. The trityl cation assay gave 5 ~mol/g loading of compound 5. The support was capped with acetic anhydride/lutidine in THF
(10% solution, 5 mL) and 1-methylimidazol in THF (16% solution, 5 mL) for 2 hours at room temperature. The support was washed with CH3CN (3 X 10 mL) and CHZCI, (1 X
10 mL). The support was treated with 20% piperidine in DMF (3 X 10 mL) to remove the Fmoc protecting group. The removal of the Fmoc group was monitored by zz~~~~s measuring UV of the solution at 302 nm. The support was washed with DMF (3 X
mL) and, then treated with TAMItA NHS ester (15 mg, 27 ~,mol) and Et3N (50 ~mol) in DMF (10 mL) for 42 hours on a shaker. The support was washed with DMF (3 X 10 mL) CH3CN (2 X 10 mL) and CH2Clz (1 X 10 mL) and dried under high vacuum for S hours.- Ninhydrin test showed less than 0.5 ~,mol/g amine left. The support was capped with acetic anhydride/lutidine in THF (10% solution, 5 mL) and 1-methylimidazol in THF (16% solution, 5 mL) for 1 hour and then washed with CH3CN (3 X 10 mL), CH2Cl2 (2 X 10 mL) and dried under high vacuum for 24 hour. The trityl cation assay showed a final loading of 4.8 ~mol/g.
Derivatization of CPG support with TAMItA dye: A mixture of CPG (500 A, 40 pmol/g amine loading, 500 mg, 20 ~,mol), compound 5 (31 mg, 22 ~mol), 1-hydroxybenzotriazol (5.9 mg, 22 ~,mol), (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexaflurophosphate (8.4 mg, 22 ~.mol), N,N
diisopropylethylamine (10.4 ~,L, 60 ~,mol) in DMF (10 mL) was shaken on a wrist action shaker for 4 hours at room temperature. The support was washed with DMF (3 X 10 mL), CH3CN (2 X 10 mL) and CHZC12 (1 X 10 mL) and dried under high vacuum overnight. The ninhydrin assay showed 4 ~,mol/g amine left. The trityl assay gave 15 ~,mol/g loading of compound 5 on CPG support. The support was capped with acetic anhydride/lutidine in THF (10% solution, 5 mL) and 1-methylimidazol in THF (16% solution, 5 mL) for hours at room temperature. The support was washed with CH3CN (3 X 10 mL) and CH.,C12 (1 X 10 mL). The support was treated with 20% piperidine in DMF (3 X
10 mL) to remove the Fmoc protecting group. Removal of the Fmoc group was monitored by measuring UV of the solution at 302 nm. The support was washed with DMF (3 X
mL). The support was then treated with TAMRA NHS ester (25 mg, 45 ~mol) and Et3N
(90 ~mol) in DMF (5 mL) for 42 hours on a shaker. The support was washed with DMF
(3 X 10 mL), CH3CN (2 X 10 mL) and CHZC12 (1 X 10 mL) and dried under high vacuum for 24 hours. Ninhydrin test showed less than 1 ~mol/g amine left. The support - was capped with acetic anhydride/lutidine in THF (10% solution, 5 mL) and 1-methylimidazol in THF (16% solution, 5 mL) for 1 hour and then washed with (3 X I 0 mL), CH,CIz (2 X 10 mL) and dried under high vacuum for 24 hours. The-trityl cation assay showed a final loading of 14 ~mol/g.
Synthesis of FAM and TAMRA Doubled Labeled Probes: Doubled dye labeled probes were synthesized by using TAMR.A labelled supports 7 and 9, DNA
FastPhosphoramidite and FAM amidite in 40 nmol scale. After completion of synthesis, supports containing probes were transferred to 4 mL glass vials and treated with a mixtl.tre of MeOHa-BuNHz:H20 (l : I :2) at 65 °C for 3 hours. Liquid was removed by a syringe and the support was washed with H~O:MeOH (3:1) and MeOH. The support was dried under vacuum and used in the hybridization assay.
3. Hybridization Ascay Usinp~jlgonucleotide Probe A 295 basepair segment of exon 3 of human beta-actin gene (nucleotides 2141-243 as disclosed in Nakajima-Iijima, S., Proc. Natl. Acad. ci U~ 82:
6133-6137 (1985) can be amplified using 50 ul reactions that contain 10 mM
Tris-HCl (pH 8.3), 50 mM KCI, 4 mM MgClz, 300 nM primer AFP [SEQ. LD. No. 7J, 300 nM
primer biotin-ARP [SEQ. LD. No. 8 with biotin attached to the 5' endJ, 200 ~eM
dATP, 200 ~M dCTP, 200 p.M dGTP, 200 p.M TTP, and 1.25 units AmpliTaq (Perkin-EImer).
The reactions are performed with (+ template) or without (no template) 20 ng human genomic DNA.
After thermal cycling at 50 °C (2 min); 95 °C (10 min); and 40 cycles of 95 °C (20 sec) followed by 60 °C (1 min), each sample is diluted by adding 200 p1 Hybridization Buffer (SX SSC, 8% (v/v) formamide, 8% (v/v) Triton X- 100). The resulting samples are transferred to a streptavidin-coated 96-well microtiter plate (Xenopore Corp., Saddle Brook, NJ) and incubated at 37 °C for 30 min in order to capture the amplified beta-actin DNA segment. Each well is then washed with 330 pl phosphate buffered saline/0.05%
TWEEN-20. Any unbiotinylated DNA strands are removed by adding 100 ~I O.I M
NaOH / 1 mM EDTA, incubating at room temperature for ~ min, and washine with ul phosphate buffered saline/0.0~% TWEEN-20. 50 ul of Hybridization Buffer containing 100 nVI ofprobe A1-26 [SEQ. LD. No. 9, nucleotides 1-26 (.~1-26), labeled *Trademark WO 96!15270 PCT/US95/14882 with reporter FAM and quencher TAMRA) is then added and incubate at 37 °C for 30 mm.
Fluorescence is then measured at 518 nm and 582 nniusing a Perkin-Elmer TaqMan LS-SOB System. The ~RQ is then calculated as described in Example 5.
The magnitude of oRQ indicates the level of hybridization of the A1-26 probe and thus is a measure of the amount of amplified beta-actin DNA segment captured in each well.
4. Hybridization Assay Using Qlieonucleotide Probe Attached To Solid SuRport Three probe/solid support combinations were examined: A1-PS: A1 [SEQ. LD.
No. 9] attached to polystyrene support; A1-CPG: Al [SEQ. LD. No. 9] attached tb glass support; and Gl-PS: G1 [SEQ. LD. No. 13] attached to polystyrene support.
All three probes have FAM attached to the 5' end of the sequence and TAMRA
attached to the 3' end. No template reactions were prepared by suspending each probe/solid support sample in 50 p1 1X PCR Buffer (10 mM Tris-HCl (pH 8.3), 50 mM
KCI, 3.5 mM MgClz). For plus template reactions, A 1-PS and A 1-CPG were suspended in 50 ~l 1X PCR Buffer+ 1 ~M A1C; G1-PS was suspended in 50 ul 1X PCR Buffer+
1 ~M G1C.
Reactions were incubated at 95 °C for 1 min, then allowed to cool slowly to room temperature. A portion of each suspension was placed on a microscope slide.
Each sample was excited with 488 nm light and a fluorescence image was captured on a CCD
array using either a 518 nm or 583 nm interference filter. The images were analyzed by Ending a peak pixel value on the 518 nm image and then finding the 583 nm value for 2~ the same pixel. Pixel values were corrected by subtracting the background readings observed with buffer. Table ~ gives the results of fluorescence measurements of the indicated probes.
*Trademark WO 96/15270 2 2 ~ 1 ~' 5 6 pGT/US95/148 Table 5.
PROBE 518 582 RQ- RQ+ 0 RQ
no +temp. no temp. +temp. -temp.
Al-PS 149 354 253 379 0.42 0.67 0.25 - -Al-CPG 494 437 1500 616 1.13 2.44 1.31 Gl-PS 75 166 178 245 0.45 0.73 0.28 5. Method For Monitoring PCR Amplification sing Oli;~~nucleotide Probe All PCR amplifications were performed in a Perkin-Elmer Thermocycler 9600 using 50 p.l reactions that contained 10 mM°Tris-HCl (pH 8.3), 50 mM
KCI, 200 ~.M
dATP, 200 p,M dCTP, 200 ~.M dGTP, 400 ~,M dUTP, 0.5 units AmpEraseTM uracil N-glycolyase (Perkin-Elmer), and 1.25 units AmpliTaqTM (Perkin-Elmer). A 295 basepair segment of exon 3 of human ~-actin gene (nucleotides 2141-2435 disclosed by Nakajima-Iijima, S., P_roc. Natl. Acad Sci SA 82: 6133-6137 (1985) was amplified using the AFP and ARP primers listed below. The amplification reactions contained 4 mM MgCl2, 20 ng human genomic DNA, 50 nM A1 or A3 probe, and 300 nM of each primer. Thermal regimen was 50 °C (2 min); 95 °C (10 min); 40 cycles of 95 °C (20 sec); 60 °C (1 min); and hold at 72 °C. A 515 basepair segment was amplified from a plasmid that consists of a segment of .1 DNA (nucleotides 32, 220-32, 747) inserted into the Sma I site of vector pUCl 19. These reactions contained 3.5 mM MgCI,, 1 ng plasmid DNA, 50 nMP2 or PS probe, 200 nM primer F 119, and 200 nM primer 8119.
The thermal regimen was 50 °C (2 min); 95 °C (10 min); 25 cycles of 95 °C (20 sec), 57 °C (1 min); and hold at 72 °C.
For each amplification reaction, 40 ~.l was transferred to an individual well of a white 96-well microtiter plate (Perkin-Elmer). Fluorescence was measured on a Perkin-Elmer TaqManTM LS-SOB System, which consists of a luminescence spectrometer with a plate reader assembly, a 485 nm excitation filter, and a 515 nm emission filter.
WO 96/15270 PCTlUS95/14882 Excitation was carried out at 488 nm using a 5 nm slit width. Emission was measured at 518 nm for 6-FAM (the reporter, or R Valve) and 582 nm for TAMR.A (the quencher, or Q value) using a 10 nm slit width. In order to determine the increase in reporter emission ' that is due to cleavage of the probe during PCR, three normalizations are applied to the raw emission data. First, emission intensity of a buffer blank is subtracted for each wavelength. Second, emission intensity of the reporter is divided by the emission intensity of the quencher to give an RQ ratio for each reaction tube. This normalizes for well-to-well variation in probe concentration and fluorescence measurement.
Finally, ~RQ is calculated by subtracting the RQ value of the no template control (RQ') from the RQ value for the complete reaction including a template (RQ~.
Three pairs of probes were tested in PCR assays. For each pair, one probe has TAMRA attached to an internal nucleotide and the other has TAMRA attached to the 3' end nucleotide. Results are shown in Table 6. For all three sets, the probe with the 3' quencher exhibits a ORQ value that is considerable higher than for the probe with the internal quencher.
Table 6.
PROBE 518 582 RQ- RQ+ ORQ
no temp. +temp. no temp. +temp.
A3-6 34.06 50.1 73.78 70.8 0.5 0.71 0.25 A3-24 58.85 202 69.66 78.8 0.8 2.57 1.72 P2-7 67.58 341 85.78 87.9 0.8 3.89 3.1 P2-27 124.6 722 152.6 118 0.8 6.1 5.28 PS-10 77.32 156 75.41 67 1 2.33 1.3 PS-28 73.23 507 106.6 96.3 0.7 5.28 4.59 WO 96/15270 ~ 7 PGT/US95/14882 Table 7. Fluorescence In Single And Double-stranded States.
Probe 518 582 RQ
ss ds ss ds ss ds - --PZ-7 63.81 84.07 96.52 142.97 0.66 0.59 _ _ P2-27 92.31 557.53 165.13 89.47 0.56 6.23 PS-10 266.30 366.37 437.97 491.00 0.61 0.75 PS-28 51.91 782.80 141.20 154.07 0.37 5.08 Al-7 18.40 60.45 105.53 218.83 0.17 0.28 Al-26 87.75 734.37 90.91 118.57 0.97 6.19 44.77 104.80 90.80 177.87 0.49 0.59 A3-24 45.57 857.57 100.15 191.43 0.46 3.47 Table 7 gives the results of fluorescence measurements of the indicated probes in single and double-stranded states. For probes having reporter and quencher at opposite ends of the oligonucleotide, hybridization caused a dramatic increase in RQ.
The foregoing description of a preferred embodiment of the invention has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise forms disclosed. Obviously, many modifications and variations will be apparent to practitioners skilled in this art. It is intended that the scope of the invention be defined by the following claims and their equivalents.
WO 96/15270 ~ ~ ~ PCT/US95/14882 SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Perkin-Elmer Corporation, Applied Biosystems Division (ii) TITLE OF INVENTION: SELF-QUENCHING FLUORESCENCE
PROBE AND METHOD
(iii) NUMBER OF SEQUENCES: 14 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: David J. Weitz, Haynes & Davis (B) STREET: 2180 Sand Hill Road, Suite 310 (C) CITY: Menlo Park (D) STATE: California (E) COUNTRY: USA
(F) ZIP: 94025-6935 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: 3.5 inch diskette (B) COMPUTER: IBM compatible (C) OPERATING SYSTEM: Microsoft Windows 3.1/DOS
5.0 (D) SOFTWARE: Wordperfect for windows 6.0, ASCII (DOS) TEXT format (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/340,558 (B) FILING DATE: 16-NOV-94 (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: David J. Weitz (B) REGISTRATION NUMBER: 38,362 (C) REFERENCE/DOCKET NUMBER: PELM4264CIP1W0 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (415) 233-0188 (B) TELEFAX: (415) 233-1129 (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
2201~~~
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
(2) INFORMATION FOR SEQ ID NO: 4 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4 (2) INFORMATION FOR SEQ ID NO: 5 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5 CGGATTTGCT GGTATCTATG ACAAGGAT 2g (2) INFORMATION FOR SEQ ID NO: 6 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6 _ (2) INFORMATION FOR SEQ ID NO: 7 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7 (2) INFORMATION FOR SEQ ID NO: 8 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8 (2) INFORMATION FOR SEQ ID NO: 9 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single zzo~7~~
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9 (2) INFORMATION FOR SEQ ID NO: 10 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single ( D ) TOPOLOGY : 1 inear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10 (2) INFORMATION FOR SEQ ID NO: 11 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11 (2) INFORMATION FOR SEQ ID NO: 12 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12 WO 96!15270 (2) INFORMATION FOR SEQ ID NO: 13 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13 (2) INFORMATION FOR SEQ ID NO: 14 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14
Claims (36)
1. 1. A method of nucleic acid amplification comprising:
performing nucleic acid amplification on a target polynucleotide using a nucleic acid polymerase having 5' to 3' nuclease activity, a primer capable of hybridizing to the target polynucleotide, and an oligonucleotide probe under amplification conditions such that the probe hybridizes to the target polynucleotide 3' relative to the primer and the probe does not hybridize with itself to form a hairpin structure, the oligonucleotide probe having at one end a fluorescent reporter and at the other end a quencher that quenches the fluorescence of the reporter molecule when both the fluorescent reporter and quencher are attached to the probe, under conditions such that digestion of the oligonucleotide probe by the polymerase during amplification is effective to separate the reporter from the quencher, whereby a fluorescence signal of the reporter is increased.
performing nucleic acid amplification on a target polynucleotide using a nucleic acid polymerase having 5' to 3' nuclease activity, a primer capable of hybridizing to the target polynucleotide, and an oligonucleotide probe under amplification conditions such that the probe hybridizes to the target polynucleotide 3' relative to the primer and the probe does not hybridize with itself to form a hairpin structure, the oligonucleotide probe having at one end a fluorescent reporter and at the other end a quencher that quenches the fluorescence of the reporter molecule when both the fluorescent reporter and quencher are attached to the probe, under conditions such that digestion of the oligonucleotide probe by the polymerase during amplification is effective to separate the reporter from the quencher, whereby a fluorescence signal of the reporter is increased.
2. The method of claim 1, wherein the polymerase is a thermostable nucleic acid polymerase.
3. The method of claim 1, wherein the reporter is a fluorescein dye and the quencher is a rhodamine dye.
4. The method of claim 3, wherein the fluorescent reporter is attached to the 5' end of the probe.
5. The method of claim 4, wherein the probe is 15 to 60 nucleotides in length.
6. The method of claim 4, wherein the probe is 18 to 30 nucleotides in length.
7. The method of claim 3, wherein the fluorescent reporter is attached to the 3' end of the probe.
8. The method of claim 7, wherein the probe is 15 to 60 nucleotides in length.
9. The method of claim 7, wherein the probe is 18 to 30 nucleotides in length.
10. The method of claim 1, wherein the fluorescent reporter is attached to the 5' end of the probe.
11. The method of claim 10, wherein the probe is 15 to 60 nucleotides in length.
12. The method of claim 10, wherein the probe is 18 to 30 nucleotides in length.
13. The method of claim 1, wherein the fluorescent reporter is attached to the 3' end of the probe.
14. The method of claim 13, wherein the probe is 15 to 60 nucleotides in length.
15. The method of claim 13, wherein the probe is 18 to 30 nucleotides in length.
16. The method of claim 1, wherein the reporter or the quencher comprises a rhodamine dye.
17. The method of claim 1, wherein the reporter or the quencher comprises a fluorescein dye.
18. The method of claim 1, wherein the reporter or quencher is selected from the group consisting of a xanthene dye and a naphthylamine dye.
19. The method of claim 1, wherein the oligonucleotide probe is not capable of extension by the nucleic acid polymerase.
20. The method of claim 19, wherein the polymerase is a thermostable nucleic acid polymerase.
21. The method of claim 19, wherein the reporter is a fluorescein dye and the quencher is a rhodamine dye.
22. The method of claim 21, wherein the fluorescent reporter is attached to the 5' end of the probe.
23. The method of claim 22, wherein the probe is 15 to 60 nucleotides in length.
24. The method of claim 22, wherein the probe is 18 to 30 nucleotides in length.
25. The method of claim 21, wherein the fluorescent reporter is attached to the 3' end of the probe.
26. The method of claim 25, wherein the probe is 15 to 60 nucleotides in length.
27. The method of claim 25, wherein the probe is 18 to 30 nucleotides in length.
28. The method of claim 19, wherein the fluorescent reporter is attached to the 5' end of the probe.
29. The method of claim 28, wherein the probe is 15 to 60 nucleotides in length.
30. The method of claim 28, wherein the probe is 18 to 30 nucleotides in length.
31. The method of claim 19, wherein the fluorescent reporter is attached to the 3' end of the probe.
32. The method of claim 31, wherein the probe is 15 to 60 nucleotides in length.
33. The method of claim 31, wherein the probe is 18 to 30 nucleotides in length.
34. The method of claim 19, wherein the fluorescent reporter or the quencher comprises a rhodamine dye.
35. The method of claim 19, wherein the fluorescent reporter or the quencher comprises a fluorescein dye.
36. The method of claim 19, wherein the reporter or quencher is selected from the group consisting of a xanthene dye and a naphthylamine dye.
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US08/340,558 US5538848A (en) | 1994-11-16 | 1994-11-16 | Method for detecting nucleic acid amplification using self-quenching fluorescence probe |
US08/340,558 | 1994-11-16 | ||
PCT/US1995/014882 WO1996015270A1 (en) | 1994-11-16 | 1995-11-15 | Self-quenching fluorescence probe and method |
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EP (2) | EP0972848A3 (en) |
JP (3) | JP4131749B2 (en) |
AT (1) | ATE198775T1 (en) |
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Families Citing this family (1090)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5837453A (en) * | 1992-05-13 | 1998-11-17 | Geron Corporation | Telomerase activity assays |
US6576419B1 (en) * | 1993-07-23 | 2003-06-10 | University Of Utah Research Foundation | Assay procedure using fluorogenic tracers |
US5804380A (en) * | 1993-11-12 | 1998-09-08 | Geron Corporation | Telomerase activity assays |
US5863726A (en) * | 1993-11-12 | 1999-01-26 | Geron Corporation | Telomerase activity assays |
US5925517A (en) * | 1993-11-12 | 1999-07-20 | The Public Health Research Institute Of The City Of New York, Inc. | Detectably labeled dual conformation oligonucleotide probes, assays and kits |
US5538848A (en) * | 1994-11-16 | 1996-07-23 | Applied Biosystems Division, Perkin-Elmer Corp. | Method for detecting nucleic acid amplification using self-quenching fluorescence probe |
US6821727B1 (en) | 1993-11-15 | 2004-11-23 | Applera Corporation | Hybridization assay using self-quenching fluorescence probe |
US6028190A (en) * | 1994-02-01 | 2000-02-22 | The Regents Of The University Of California | Probes labeled with energy transfer coupled dyes |
US5547861A (en) | 1994-04-18 | 1996-08-20 | Becton, Dickinson And Company | Detection of nucleic acid amplification |
JP2909216B2 (en) * | 1994-04-29 | 1999-06-23 | パーキン‐エルマー コーポレイション | Real-time detection device for nucleic acid amplification products |
US20070269799A9 (en) * | 1994-06-22 | 2007-11-22 | Zhang David Y | Nucleic acid amplification methods |
US6787304B1 (en) | 1994-12-28 | 2004-09-07 | Georgetown University | Fluorometric assay for detecting nucleic acid cleavage |
US20030165908A1 (en) * | 1994-12-30 | 2003-09-04 | Georgetown University | Fluorometric assay for detecting nucleic acid cleavage |
US6312894B1 (en) * | 1995-04-03 | 2001-11-06 | Epoch Pharmaceuticals, Inc. | Hybridization and mismatch discrimination using oligonucleotides conjugated to minor groove binders |
US5801155A (en) * | 1995-04-03 | 1998-09-01 | Epoch Pharmaceuticals, Inc. | Covalently linked oligonucleotide minor grove binder conjugates |
AU693023B2 (en) * | 1995-05-05 | 1998-06-18 | Applied Biosystems, Llc | Methods and reagents for combined PCR amplification and hybridization probing assay |
WO1997015687A1 (en) * | 1995-06-07 | 1997-05-01 | Geron Corporation | Telomerase activity assays |
KR100463475B1 (en) * | 1995-06-08 | 2005-06-22 | 로셰 디아그노스틱스 게엠베하 | Magnetic Pigment |
CA2225531A1 (en) * | 1995-06-23 | 1997-01-09 | Baylor College Of Medicine | Alternative dye-labeled primers, ribonucleotides, deoxyribonucleotides, and dideoxyribonucleotides for automated dna analysis and homogeneous amplification/detection assays |
US5994063A (en) * | 1995-06-23 | 1999-11-30 | Metzker; Michael L. | Substituted 4,4-difluoro-4-bora-3A,4A-diaza-s-indacene compounds for homogenous amplification/detection assays |
US20020022261A1 (en) * | 1995-06-29 | 2002-02-21 | Anderson Rolfe C. | Miniaturized genetic analysis systems and methods |
US5945283A (en) * | 1995-12-18 | 1999-08-31 | Washington University | Methods and kits for nucleic acid analysis using fluorescence resonance energy transfer |
US5721123A (en) * | 1996-01-05 | 1998-02-24 | Microfab Technology, Inc. | Methods and apparatus for direct heating of biological material |
AU2735797A (en) * | 1996-04-12 | 1998-03-26 | Public Health Research Institute Of The City Of New York, Inc., The | Detection probes, kits and assays |
DE69738687D1 (en) * | 1996-04-12 | 2008-06-26 | Phri Properties Inc | PROBES, KITS AND ASSAYS |
US5955268A (en) * | 1996-04-26 | 1999-09-21 | Abbott Laboratories | Method and reagent for detecting multiple nucleic acid sequences in a test sample |
DE69736475T2 (en) * | 1996-05-02 | 2007-08-16 | Applera Corp., Foster City | QUANTIFYING RNA TRANSCRIPTS USING GENOMIC DNA AS THE INTERNAL STANDARD OF THE AMPLIFICATION REACTION |
US5736333A (en) * | 1996-06-04 | 1998-04-07 | The Perkin-Elmer Corporation | Passive internal references for the detection of nucleic acid amplification products |
JP2000514307A (en) * | 1996-07-16 | 2000-10-31 | ジェン―プローブ・インコーポレーテッド | Methods for detecting and amplifying nucleic acid sequences using modified oligonucleotides with increased target specific T <M> |
US7070925B1 (en) | 1996-07-16 | 2006-07-04 | Gen-Probe Incorporated | Method for determining the presence of an RNA analyte in a sample using a modified oligonucleotide probe |
US5853990A (en) * | 1996-07-26 | 1998-12-29 | Edward E. Winger | Real time homogeneous nucleotide assay |
US5691145A (en) * | 1996-08-27 | 1997-11-25 | Becton, Dickinson And Company | Detection of nucleic acids using G-quartets |
US5853992A (en) * | 1996-10-04 | 1998-12-29 | The Regents Of The University Of California | Cyanine dyes with high-absorbance cross section as donor chromophores in energy transfer labels |
GB2333597B (en) | 1996-10-29 | 2001-04-18 | Univ Nebraska At Lincoln | Method for detecting point mutations in dna utilizing fluorescence energy transfer |
US6017702A (en) * | 1996-12-05 | 2000-01-25 | The Perkin-Elmer Corporation | Chain-termination type nucleic acid sequencing method including 2'-deoxyuridine-5'-triphosphate |
US6312886B1 (en) | 1996-12-06 | 2001-11-06 | The Secretary Of State For Defence In Her Brittanic Majesty's Government Of The United Kingdom Of Great Britain And Northern Ireland | Reaction vessels |
GB9716052D0 (en) * | 1996-12-06 | 1997-10-01 | Secr Defence | Reaction vessels |
DE69825601T2 (en) | 1997-02-12 | 2005-04-28 | Chan, Eugene Y, Brookline | METHOD FOR THE ANALYSIS OF POLYMERS |
US6403311B1 (en) | 1997-02-12 | 2002-06-11 | Us Genomics | Methods of analyzing polymers using ordered label strategies |
AU6822598A (en) * | 1997-02-24 | 1998-09-09 | Georgia Tech Research Corporation | Method for determining a nucleic acid |
US6117973A (en) * | 1997-02-24 | 2000-09-12 | Georgia Tech Research Corp. | PNA monomers with electron donor or acceptor |
US6251591B1 (en) | 1997-02-27 | 2001-06-26 | Lorne Park Research, Inc. | Quantitative method for detecting nucleotide concentration |
US6060242A (en) * | 1997-02-27 | 2000-05-09 | Lorne Park Research, Inc. | PNA diagnostic methods |
US6046004A (en) * | 1997-02-27 | 2000-04-04 | Lorne Park Research, Inc. | Solution hybridization of nucleic acids with antisense probes having modified backbones |
US5846729A (en) * | 1997-02-27 | 1998-12-08 | Lorne Park Research, Inc. | Assaying nucleotides in solution using a fluorescent intensity quenching effect |
EP1666609B1 (en) * | 1997-02-28 | 2012-09-26 | Quest Diagnostics Investments Incorporated | Fluorescence energy transfer by competitive hybridization |
JP3016759B2 (en) * | 1997-02-28 | 2000-03-06 | スミスクライン・ビーチャム・コーポレイション | Fluorescent energy transfer by competitive hybridization |
US7803528B1 (en) | 1997-02-28 | 2010-09-28 | Quest Diagnostics Incorporated | Fluorescence energy transfer by competitive hybridization |
US6143496A (en) * | 1997-04-17 | 2000-11-07 | Cytonix Corporation | Method of sampling, amplifying and quantifying segment of nucleic acid, polymerase chain reaction assembly having nanoliter-sized sample chambers, and method of filling assembly |
US5846726A (en) * | 1997-05-13 | 1998-12-08 | Becton, Dickinson And Company | Detection of nucleic acids by fluorescence quenching |
US6465175B2 (en) | 1997-09-04 | 2002-10-15 | Bayer Corporation | Oligonucleotide probes bearing quenchable fluorescent labels, and methods of use thereof |
US6342350B1 (en) * | 1997-09-05 | 2002-01-29 | The General Hospital Corporation | Alpha-2-macroglobulin diagnostic test |
AU743011B2 (en) | 1997-09-12 | 2002-01-17 | Phri Properties, Inc. | Non-competitive co-amplification methods |
CA2303945A1 (en) * | 1997-09-18 | 1999-03-25 | Erasmus Universiteit Rotterdam | Detection of minimal residual disease in lymphoid malignancies |
US5935791A (en) * | 1997-09-23 | 1999-08-10 | Becton, Dickinson And Company | Detection of nucleic acids by fluorescence quenching |
DE19743518A1 (en) * | 1997-10-01 | 1999-04-15 | Roche Diagnostics Gmbh | Automated, universally applicable sample preparation method |
US6485901B1 (en) | 1997-10-27 | 2002-11-26 | Boston Probes, Inc. | Methods, kits and compositions pertaining to linear beacons |
WO1999022018A2 (en) | 1997-10-27 | 1999-05-06 | Boston Probes, Inc. | Methods, kits and compositions pertaining to pna molecular beacons |
CA2308762A1 (en) * | 1997-11-04 | 1999-05-14 | Roche Diagnostics Gmbh | Specific and sensitive nucleic acid detection method |
US6294326B1 (en) | 1997-11-07 | 2001-09-25 | Abbott Laboratories | Analyte detection process using dual labeled probes |
US6583168B1 (en) | 1997-11-25 | 2003-06-24 | Applera Corporation | Sulfonated diarylrhodamine dyes |
DE19752898A1 (en) * | 1997-11-28 | 1999-08-05 | Centeon Pharma Gmbh | Method for the detection of high concentrations of four in blood plasma and / or blood serum by means of the polymerase chain reaction |
US6893877B2 (en) | 1998-01-12 | 2005-05-17 | Massachusetts Institute Of Technology | Methods for screening substances in a microwell array |
US6289229B1 (en) | 1998-01-20 | 2001-09-11 | Scimed Life Systems, Inc. | Readable probe array for in vivo use |
US6080868A (en) * | 1998-01-23 | 2000-06-27 | The Perkin-Elmer Corporation | Nitro-substituted non-fluorescent asymmetric cyanine dye compounds |
WO1999040226A2 (en) * | 1998-02-04 | 1999-08-12 | Perkin-Elmer Corporation | Determination of a genotype of an amplification product at multiple allelic sites |
WO1999044045A1 (en) * | 1998-02-27 | 1999-09-02 | Massachusetts Institute Of Technology | Single molecule detection with surface-enhanced raman scattering and applications in dna or rna sequencing |
US6235480B1 (en) | 1998-03-13 | 2001-05-22 | Promega Corporation | Detection of nucleic acid hybrids |
US7090975B2 (en) | 1998-03-13 | 2006-08-15 | Promega Corporation | Pyrophosphorolysis and incorporation of nucleotide method for nucleic acid detection |
US6277578B1 (en) | 1998-03-13 | 2001-08-21 | Promega Corporation | Deploymerization method for nucleic acid detection of an amplified nucleic acid target |
US6312902B1 (en) | 1998-03-13 | 2001-11-06 | Promega Corporation | Nucleic acid detection |
US6270974B1 (en) | 1998-03-13 | 2001-08-07 | Promega Corporation | Exogenous nucleic acid detection |
US6268146B1 (en) | 1998-03-13 | 2001-07-31 | Promega Corporation | Analytical methods and materials for nucleic acid detection |
US6270973B1 (en) | 1998-03-13 | 2001-08-07 | Promega Corporation | Multiplex method for nucleic acid detection |
US6391551B1 (en) | 1998-03-13 | 2002-05-21 | Promega Corporation | Detection of nucleic acid hybrids |
US6703211B1 (en) | 1998-03-13 | 2004-03-09 | Promega Corporation | Cellular detection by providing high energy phosphate donor other than ADP to produce ATP |
DE19811730A1 (en) * | 1998-03-18 | 1999-09-23 | November Ag Molekulare Medizin | Identifying marker that indicates presence of immobilized nucleic acid using fluorophore-labeled detection agent bound to solid phase |
DE19811731A1 (en) * | 1998-03-18 | 1999-09-23 | November Ag Molekulare Medizin | Detecting nucleic acid by amplification, with use of third, immobilized primer |
DE19811729C2 (en) * | 1998-03-18 | 2000-05-18 | November Ag Molekulare Medizin | Method and device for detecting a nucleotide sequence |
US6361942B1 (en) * | 1998-03-24 | 2002-03-26 | Boston Probes, Inc. | Method, kits and compositions pertaining to detection complexes |
US5952202A (en) * | 1998-03-26 | 1999-09-14 | The Perkin Elmer Corporation | Methods using exogenous, internal controls and analogue blocks during nucleic acid amplification |
US6387652B1 (en) | 1998-04-15 | 2002-05-14 | U.S. Environmental Protection Agency | Method of identifying and quantifying specific fungi and bacteria |
US6287772B1 (en) * | 1998-04-29 | 2001-09-11 | Boston Probes, Inc. | Methods, kits and compositions for detecting and quantitating target sequences |
DE69936897T2 (en) * | 1998-05-01 | 2008-05-15 | Gen-Probe Inc., San Diego | Incubator for automatic analyzer |
AU3979099A (en) | 1998-05-11 | 1999-11-29 | Philadelphia Health & Education Corporation | (mct-1), a human oncogene |
US6255050B1 (en) | 1998-05-22 | 2001-07-03 | Lorne Park Research, Inc. | Dynamic hybridization system |
GB9812768D0 (en) * | 1998-06-13 | 1998-08-12 | Zeneca Ltd | Methods |
ATE440963T1 (en) * | 1998-07-02 | 2009-09-15 | Gen Probe Inc | MOLECULAR TORCHES |
GB9815933D0 (en) * | 1998-07-23 | 1998-09-23 | Secr Defence | Detection method |
US6037130A (en) * | 1998-07-28 | 2000-03-14 | The Public Health Institute Of The City Of New York, Inc. | Wavelength-shifting probes and primers and their use in assays and kits |
US6210896B1 (en) | 1998-08-13 | 2001-04-03 | Us Genomics | Molecular motors |
US6500650B1 (en) * | 1998-10-01 | 2002-12-31 | Variagenics, Inc. | Method for identifying polymorphisms |
US6153411A (en) * | 1998-10-30 | 2000-11-28 | American Water Works Company, Inc. | Methods and kits for detection of Cryptosporidium parvum using immunomagnetic separation and amplification |
US6245518B1 (en) * | 1998-12-11 | 2001-06-12 | Hyseq, Inc. | Polynucleotide arrays and methods of making and using the same |
WO2000036152A1 (en) | 1998-12-14 | 2000-06-22 | Li-Cor, Inc. | A system and methods for nucleic acid sequencing of single molecules by polymerase synthesis |
US6420115B1 (en) | 1999-12-21 | 2002-07-16 | Ingeneus Corporation | Cation mediated triplex hybridization assay |
US6403313B1 (en) | 1999-12-21 | 2002-06-11 | Ingeneus Corporation | Fluorescent intensity assay for duplex and triplex nucleic acid hybridization solution utilizing fluorescent intercalators |
US6858390B2 (en) | 1998-12-31 | 2005-02-22 | Ingeneus Corporation | Aptamers containing sequences of nucleic acid or nucleic acid analogues bound homologously, or in novel complexes |
US6656692B2 (en) | 1999-12-21 | 2003-12-02 | Ingeneus Corporation | Parallel or antiparallel, homologous or complementary binding of nucleic acids or analogues thereof to form duplex, triplex or quadruplex complexes |
US6432642B1 (en) * | 1999-01-15 | 2002-08-13 | Pe Corporation (Ny) | Binary probe and clamp composition and methods for a target hybridization detection |
US6951722B2 (en) * | 1999-03-19 | 2005-10-04 | Takara Bio Inc. | Method for amplifying nucleic acid sequence |
CN1348396A (en) | 1999-03-19 | 2002-05-08 | 金克克国际有限公司 | Multi-through hole testing plate for high throughput screening |
US6627400B1 (en) | 1999-04-30 | 2003-09-30 | Aclara Biosciences, Inc. | Multiplexed measurement of membrane protein populations |
US20040248150A1 (en) * | 1999-04-02 | 2004-12-09 | Sharat Singh | Methods employing oligonucleotide-binding e-tag probes |
US6322980B1 (en) * | 1999-04-30 | 2001-11-27 | Aclara Biosciences, Inc. | Single nucleotide detection using degradation of a fluorescent sequence |
US6573047B1 (en) | 1999-04-13 | 2003-06-03 | Dna Sciences, Inc. | Detection of nucleotide sequence variation through fluorescence resonance energy transfer label generation |
CA2304260C (en) * | 1999-04-20 | 2009-03-24 | Japan Bioindustry Association | Method for determining a concentration of target nucleic acid molecules, nucleic acid probes for the method and method for analyzing data obtained by the method |
US20030235832A1 (en) * | 2000-06-21 | 2003-12-25 | Ahmed Chenna | Multiplexed analysis by chromatographic separation of molecular tags |
US7001725B2 (en) | 1999-04-30 | 2006-02-21 | Aclara Biosciences, Inc. | Kits employing generalized target-binding e-tag probes |
US6673550B2 (en) | 1999-04-30 | 2004-01-06 | Aclara Biosciences, Inc. | Electrophoretic tag reagents comprising fluorescent compounds |
US7037654B2 (en) | 1999-04-30 | 2006-05-02 | Aclara Biosciences, Inc. | Methods and compositions for enhancing detection in determinations employing cleavable electrophoretic tag reagents |
US6331393B1 (en) * | 1999-05-14 | 2001-12-18 | University Of Southern California | Process for high-throughput DNA methylation analysis |
US6180349B1 (en) | 1999-05-18 | 2001-01-30 | The Regents Of The University Of California | Quantitative PCR method to enumerate DNA copy number |
US7056661B2 (en) | 1999-05-19 | 2006-06-06 | Cornell Research Foundation, Inc. | Method for sequencing nucleic acid molecules |
EP1681357A3 (en) * | 1999-05-19 | 2006-07-26 | Cornell Research Foundation, Inc. | Method for sequencing nucleic acid molecules |
US6277607B1 (en) | 1999-05-24 | 2001-08-21 | Sanjay Tyagi | High specificity primers, amplification methods and kits |
US7097973B1 (en) | 1999-06-14 | 2006-08-29 | Alpha Mos | Method for monitoring molecular species within a medium |
WO2000077248A1 (en) * | 1999-06-15 | 2000-12-21 | november Aktiengesellschaft Gesellschaft für Molekulare Medizin | Device and method for storing information |
WO2000079009A2 (en) * | 1999-06-22 | 2000-12-28 | Invitrogen Corporation | Improved primers and methods for the detection and discrimination of nucleic acids |
US6645733B1 (en) | 1999-06-25 | 2003-11-11 | Ingeneus Corporation | Fluorescent intensity method for assaying binding between proteins or peptides |
US6258593B1 (en) * | 1999-06-30 | 2001-07-10 | Agilent Technologies Inc. | Apparatus for conducting chemical or biochemical reactions on a solid surface within an enclosed chamber |
US6830902B1 (en) * | 1999-07-02 | 2004-12-14 | Invitrogen Corporation | Compositions and methods for enhanced sensitivity and specificity of nucleic acid synthesis |
EP1210358A4 (en) * | 1999-08-13 | 2005-01-05 | Univ Brandeis | Detection of nucleic acids |
DK1204856T3 (en) † | 1999-08-20 | 2005-08-08 | Diagnostische Forsch Stiftung | Method for Determining Substances by the Evanescence Field Method |
CA2385658A1 (en) * | 1999-09-22 | 2001-03-29 | Wakunaga Pharmaceutical Co., Ltd. | Hybridization self-recognition probe |
ATE237399T1 (en) | 1999-09-29 | 2003-05-15 | Tecan Trading Ag | THERMOCYCLER AND LIFTING ELEMENT FOR MICROTITER PLATE |
US6272939B1 (en) | 1999-10-15 | 2001-08-14 | Applera Corporation | System and method for filling a substrate with a liquid sample |
US6893819B1 (en) * | 2000-11-21 | 2005-05-17 | Stratagene California | Methods for detection of a nucleic acid by sequential amplification |
US7585632B2 (en) * | 1999-10-29 | 2009-09-08 | Hologic, Inc. | Compositions and methods for the detection of a nucleic acid using a cleavage reaction |
US7381532B2 (en) * | 1999-10-29 | 2008-06-03 | Stratagene California | Compositions and methods for the detection of a nucleic acid using a cleavage reaction |
US7534568B2 (en) | 1999-10-29 | 2009-05-19 | Hologic Inc. | Methods for detection of a target nucleic acid by forming a cleavage structure with a cleavage resistant probe |
US7118860B2 (en) * | 1999-10-29 | 2006-10-10 | Stratagene California | Methods for detection of a target nucleic acid by capture |
US7838225B2 (en) * | 1999-10-29 | 2010-11-23 | Hologic, Inc. | Methods for detection of a target nucleic acid by forming a cleavage structure using a reverse transcriptase |
US7824859B2 (en) * | 1999-10-29 | 2010-11-02 | Cytyc Corporation | Methods for detection of a target nucleic acid by forming a cleavage structure using an RNA polymerase |
DK1232502T3 (en) * | 1999-11-17 | 2006-05-22 | Roche Diagnostics Gmbh | Magnetic glass particles, processes for their manufacture and their applications |
WO2001038585A2 (en) | 1999-11-24 | 2001-05-31 | The Regents Of The University Of California | Polymer arrays and methods of using labeled probe molecules to identify and quantify target molecule expression |
US6458544B1 (en) | 1999-12-02 | 2002-10-01 | Dna Sciences, Inc. | Methods for determining single nucleotide variations and genotyping |
US6274321B1 (en) * | 1999-12-03 | 2001-08-14 | The Regents Of The University Of California | High throughput functional screening of cDNAs |
US6727356B1 (en) * | 1999-12-08 | 2004-04-27 | Epoch Pharmaceuticals, Inc. | Fluorescent quenching detection reagents and methods |
US20040081959A9 (en) * | 1999-12-08 | 2004-04-29 | Epoch Biosciences, Inc. | Fluorescent quenching detection reagents and methods |
GB2359625B (en) | 1999-12-10 | 2004-10-20 | Molecular Light Tech Res Ltd | Monitoring oligonucleotide binding process using chemiluminescence quenching |
US6924108B2 (en) | 1999-12-21 | 2005-08-02 | Ingeneus Corporation | Nucleic acid binding enhancement by conjugation with nucleotides, nucleosides, bases and/or their analogues |
US6911536B1 (en) | 1999-12-21 | 2005-06-28 | Ingeneus Corporation | Triplex and quadruplex catalytic hybridization |
US7309569B2 (en) | 1999-12-21 | 2007-12-18 | Ingeneus, Inc. | Parallel or antiparallel, homologous or complementary binding of nucleic acids or analogues thereof to form duplex, triplex or quadruplex complexes |
US20030181412A1 (en) * | 1999-12-21 | 2003-09-25 | Ingeneus Corporation | Method for modifying transcription and/or translation in an organism for therapeutic, prophylactic and/or analytic uses |
US7052844B2 (en) * | 1999-12-21 | 2006-05-30 | Ingeneus, Inc. | Purification of DS-DNA using heteropolymeric capture probes and a triplex, quadruplex or homologous duplex binding mechanism |
US6927027B2 (en) | 1999-12-21 | 2005-08-09 | Ingeneus Corporation | Nucleic acid multiplex formation |
US6982147B2 (en) * | 2000-01-24 | 2006-01-03 | Ingeneus Corporation | Apparatus for assaying biopolymer binding by means of multiple measurements under varied conditions |
US20030170659A1 (en) * | 2000-01-24 | 2003-09-11 | Ingeneus Corporation | Electrical treatment of binding media to encourage, discourage and/or study biopolymer binding |
US6265170B1 (en) | 2000-01-24 | 2001-07-24 | Ingeneus Corporation | Homogenous assay of duplex of triplex hybridization by means of multiple measurements under varied conditions |
US7220541B2 (en) * | 2000-01-24 | 2007-05-22 | Ingeneus, Inc. | Homogeneous assay of biopolymer binding by means of multiple measurements under varied conditions |
US6613524B1 (en) | 2000-01-24 | 2003-09-02 | Ingeneus Corporation | Amperometric affinity assay and electrically stimulated complexes of nucleic acids |
US7169355B1 (en) | 2000-02-02 | 2007-01-30 | Applera Corporation | Apparatus and method for ejecting sample well trays |
US6221604B1 (en) | 2000-02-07 | 2001-04-24 | Pe Corporation | Electron-deficient nitrogen heterocycle-substituted fluorescein dyes |
US6418383B1 (en) * | 2000-02-11 | 2002-07-09 | Perkinelmer Instruments Llc | Method and apparatus for iterative spectral compensation |
FI20000333A0 (en) * | 2000-02-16 | 2000-02-16 | Jussi Nurmi | A homogeneous method for detecting a polynucleotide |
US20020151040A1 (en) | 2000-02-18 | 2002-10-17 | Matthew O' Keefe | Apparatus and methods for parallel processing of microvolume liquid reactions |
FR2805348B1 (en) * | 2000-02-23 | 2002-07-12 | Commissariat Energie Atomique | BIOLOGICAL TARGET ANALYSIS USING A BIOCHIP COMPRISING A FLUORESCENT MARKER |
EP1254230A2 (en) | 2000-02-29 | 2002-11-06 | Millennium Pharmaceuticals, Inc. | 1983, 52881, 2398, 45449, 50289, and 52872, g protein-coupled receptors and uses therefor |
GB0005281D0 (en) * | 2000-03-07 | 2000-04-26 | Secr Defence | Analytical method |
US7205104B2 (en) * | 2000-03-24 | 2007-04-17 | Eppendorf Array Technologies Sa (Eat) | Identification of biological (micro) organisms by detection of their homologous nucleotide sequences on arrays |
US7202026B2 (en) * | 2000-03-24 | 2007-04-10 | Eppendorf Array Technologies Sa (Eat) | Identification of a large number of biological (micro)organisms groups at different levels by their detection on a same array |
US7875442B2 (en) * | 2000-03-24 | 2011-01-25 | Eppendorf Array Technologies | Identification and quantification of a plurality of biological (micro)organisms or their components |
US20080085515A1 (en) * | 2000-03-24 | 2008-04-10 | Eppendorf Array Technologies Sa (Eat) | Identification of multiple biological (micro) organisms by detection of their nucleotide sequences on arrays |
US7829313B2 (en) * | 2000-03-24 | 2010-11-09 | Eppendorf Array Technologies | Identification and quantification of a plurality of biological (micro)organisms or their components |
DE60111218T2 (en) * | 2000-03-27 | 2006-03-23 | Applera Corp., Foster City | IMPROVED INVASION ASSAY |
NZ521593A (en) * | 2000-03-29 | 2004-11-26 | Lgc Ltd | Hybridisation beacon and method of rapid sequence detection and discrimination |
US7998673B2 (en) * | 2000-03-29 | 2011-08-16 | Lgc Limited | Hybridisation beacon and method of rapid sequence detection and discrimination |
US20030207300A1 (en) * | 2000-04-28 | 2003-11-06 | Matray Tracy J. | Multiplex analytical platform using molecular tags |
US7771929B2 (en) * | 2000-04-28 | 2010-08-10 | Monogram Biosciences, Inc. | Tag library compounds, compositions, kits and methods of use |
US7537938B2 (en) * | 2000-04-28 | 2009-05-26 | Monogram Biosciences, Inc. | Biomarker detection in circulating cells |
US7160735B2 (en) * | 2000-04-28 | 2007-01-09 | Monogram Biosciences, Inc. | Tagged microparticle compositions and methods |
US7019129B1 (en) * | 2000-05-09 | 2006-03-28 | Biosearch Technologies, Inc. | Dark quenchers for donor-acceptor energy transfer |
US6686188B2 (en) * | 2000-05-26 | 2004-02-03 | Amersham Plc | Polynucleotide encoding a human myosin-like polypeptide expressed predominantly in heart and muscle |
US6656700B2 (en) | 2000-05-26 | 2003-12-02 | Amersham Plc | Isoforms of human pregnancy-associated protein-E |
US6355433B1 (en) | 2000-06-02 | 2002-03-12 | Dna Sciences, Inc. | Determination of nucleotide sequence variations through limited primer extension |
US6605451B1 (en) | 2000-06-06 | 2003-08-12 | Xtrana, Inc. | Methods and devices for multiplexing amplification reactions |
EP2351853A1 (en) | 2000-06-06 | 2011-08-03 | Life Technologies Corporation | Method and devices for multiplexing amplification reactions |
US7087414B2 (en) | 2000-06-06 | 2006-08-08 | Applera Corporation | Methods and devices for multiplexing amplification reactions |
US6887664B2 (en) | 2000-06-06 | 2005-05-03 | Applera Corporation | Asynchronous primed PCR |
US6869764B2 (en) | 2000-06-07 | 2005-03-22 | L--Cor, Inc. | Nucleic acid sequencing using charge-switch nucleotides |
US6936702B2 (en) | 2000-06-07 | 2005-08-30 | Li-Cor, Inc. | Charge-switch nucleotides |
EP1164201A1 (en) * | 2000-06-14 | 2001-12-19 | Facultés Universitaires Notre-Dame de la Paix | Reverse detection for identification and/or quantification of nucleotide target sequences on biochips |
US6719949B1 (en) | 2000-06-29 | 2004-04-13 | Applera Corporation | Apparatus and method for transporting sample well trays |
EP1930447A3 (en) * | 2000-07-11 | 2008-06-25 | Kirk Hogan | Methods and compositions for perioperative genomic profiling |
US6984522B2 (en) * | 2000-08-03 | 2006-01-10 | Regents Of The University Of Michigan | Isolation and use of solid tumor stem cells |
US8044259B2 (en) | 2000-08-03 | 2011-10-25 | The Regents Of The University Of Michigan | Determining the capability of a test compound to affect solid tumor stem cells |
US20080194022A1 (en) * | 2000-08-03 | 2008-08-14 | Clarke Michael F | Isolation and use of solid tumor stem cells |
EP1307592B1 (en) | 2000-08-11 | 2006-07-05 | University of Utah Research Foundation | Single-labeled oligonucleotide probes |
US7198924B2 (en) | 2000-12-11 | 2007-04-03 | Invitrogen Corporation | Methods and compositions for synthesis of nucleic acid molecules using multiple recognition sites |
JP4128074B2 (en) * | 2000-08-23 | 2008-07-30 | タカラバイオ株式会社 | Nucleic acid amplification method |
US6358679B1 (en) * | 2000-08-24 | 2002-03-19 | Pe Corporation (Ny) | Methods for external controls for nucleic acid amplification |
US20020123474A1 (en) * | 2000-10-04 | 2002-09-05 | Shannon Mark E. | Human GTP-Rho binding protein2 |
IL155450A0 (en) * | 2000-10-20 | 2003-11-23 | Expression Diagnostics Inc | Leukocyte expression profiling |
US7060442B2 (en) * | 2000-10-30 | 2006-06-13 | Regents Of The University Of Michigan | Modulators on Nod2 signaling |
JP2004515229A (en) * | 2000-11-27 | 2004-05-27 | スローン−ケッタリング インスティテュート フォー キャンサー リサーチ | Methods of using fluorescent energy conversion probes to detect nucleic acid cleavage |
US7037652B2 (en) | 2000-11-28 | 2006-05-02 | Wyeth | Expression analysis of KIAA nucleic acids and polypeptides useful in the diagnosis and treatment of prostate cancer |
EP2316976A1 (en) | 2000-11-28 | 2011-05-04 | Wyeth LLC | Expression analysis of FKBP nucleic acids and polypeptides useful in the diagnosis and treatment of prostate cancer |
US20080261220A1 (en) * | 2000-11-30 | 2008-10-23 | Third Wave Technologies, Inc. | Nucleic Acid Detection Assays |
US7211414B2 (en) * | 2000-12-01 | 2007-05-01 | Visigen Biotechnologies, Inc. | Enzymatic nucleic acid synthesis: compositions and methods for altering monomer incorporation fidelity |
US20050267061A1 (en) * | 2004-04-08 | 2005-12-01 | Sangamo Biosciences, Inc. | Methods and compositions for treating neuropathic and neurodegenerative conditions |
DE60236068D1 (en) * | 2001-01-31 | 2010-06-02 | Mayo Foundation | PROOF OF HERPEX SIMPLEX VIRUS |
GB0105789D0 (en) * | 2001-03-08 | 2001-04-25 | Expresson Biosystems Ltd | Detecting interactions between oligonucleotides and RNA using fluorescence resonance energy transfer (FRET) |
US7267945B2 (en) * | 2001-03-26 | 2007-09-11 | Applera Corporation | Methods of determining the presence of polynucleotides employing amplification |
WO2002077282A1 (en) * | 2001-03-27 | 2002-10-03 | International Reagents Corporation | Method of detecting gene |
HUP0501021A3 (en) | 2001-05-21 | 2006-06-28 | Aclara Biosciences Inc Mountai | Methods and compositions for analyzing proteins |
WO2002095356A2 (en) * | 2001-05-21 | 2002-11-28 | Aclara Biosciences, Inc. | Methods and compositions for analyzing proteins |
AU2002344221A1 (en) * | 2001-05-26 | 2002-12-09 | Aclara Biosciences, Inc. | Catalytic amplification of multiplexed assay signals |
US20050261486A1 (en) * | 2001-05-31 | 2005-11-24 | Q-Rna | Compositions and methods for binding agglomeration proteins |
US20040005543A1 (en) * | 2002-01-18 | 2004-01-08 | Abraham Grossman | Compositions and methods for binding agglomeration proteins |
US6770440B1 (en) * | 2001-05-31 | 2004-08-03 | The United States Of America As Represented By The Secretary Of Agriculture | Polymerase chain reaction assay for the detection of Toxoplasma gondii |
US6534646B2 (en) | 2001-06-04 | 2003-03-18 | Barrskogen, Inc. | Oligonucleotide labeling reagents |
US7235358B2 (en) | 2001-06-08 | 2007-06-26 | Expression Diagnostics, Inc. | Methods and compositions for diagnosing and monitoring transplant rejection |
US7026121B1 (en) | 2001-06-08 | 2006-04-11 | Expression Diagnostics, Inc. | Methods and compositions for diagnosing and monitoring transplant rejection |
US6905827B2 (en) * | 2001-06-08 | 2005-06-14 | Expression Diagnostics, Inc. | Methods and compositions for diagnosing or monitoring auto immune and chronic inflammatory diseases |
US7297494B2 (en) * | 2001-06-25 | 2007-11-20 | Georgia Tech Research Corporation | Activatable probes and methods for in vivo gene detection |
US7081336B2 (en) * | 2001-06-25 | 2006-07-25 | Georgia Tech Research Corporation | Dual resonance energy transfer nucleic acid probes |
BRPI0211191B8 (en) * | 2001-06-28 | 2021-07-27 | Chiron Corp | nucleic acid-based assays to accurately diagnose parvovirus b19 infection and initiators and markers for use in these assays |
KR100439168B1 (en) * | 2001-07-02 | 2004-07-05 | (주)넥스젠 | DNA Amplification Kits for Detecting Genetically-Modified Plants Quantitatively and Methods Using the Same |
US6514750B2 (en) * | 2001-07-03 | 2003-02-04 | Pe Corporation (Ny) | PCR sample handling device |
GB0118758D0 (en) * | 2001-08-01 | 2001-09-26 | Sybesma Wilbert F H | Chimeric primed based real time rt-pcr for qualification of rna or dna templates in a crude sample |
US20040078837A1 (en) * | 2001-08-02 | 2004-04-22 | Shannon Mark E. | Four human zinc-finger-containing proteins: MDZ3, MDZ4, MDZ7 and MDZ12 |
US7229774B2 (en) | 2001-08-02 | 2007-06-12 | Regents Of The University Of Michigan | Expression profile of prostate cancer |
PT1421200E (en) * | 2001-08-23 | 2007-02-28 | Merck & Co Inc | Fluorescent multiplex hpv pcr assays using multiple fluorophores |
US20030054413A1 (en) * | 2001-08-23 | 2003-03-20 | Sriram Kumaraswamy | Bio-sensing platforms for detection and quantitation of biological molecules |
US20030082547A1 (en) | 2001-08-27 | 2003-05-01 | Ewing Gregory J. | Non-fluorescent quencher compounds and biomolecular assays |
US20030108972A1 (en) * | 2001-12-06 | 2003-06-12 | Zweig Stephen Eliot | Tethered receptor-ligand reagent and assay |
US20050042629A1 (en) | 2002-09-13 | 2005-02-24 | Applera Corporation | Thermus scotoductus nucleic acid polymerases |
US7108975B2 (en) * | 2001-09-21 | 2006-09-19 | Regents Of The University Of Michigan | Atlastin |
US7582425B2 (en) * | 2001-09-21 | 2009-09-01 | The Regents Of The University Of Michigan | Atlastin |
US6593091B2 (en) | 2001-09-24 | 2003-07-15 | Beckman Coulter, Inc. | Oligonucleotide probes for detecting nucleic acids through changes in flourescence resonance energy transfer |
US6744502B2 (en) * | 2001-09-28 | 2004-06-01 | Pe Corporation (Ny) | Shaped illumination geometry and intensity using a diffractive optical element |
US20040014067A1 (en) * | 2001-10-12 | 2004-01-22 | Third Wave Technologies, Inc. | Amplification methods and compositions |
US6942836B2 (en) * | 2001-10-16 | 2005-09-13 | Applera Corporation | System for filling substrate chambers with liquid |
EP1442142A4 (en) * | 2001-10-19 | 2006-11-15 | Proligo Llc | Nucleic acid probes and methods to detect and/or quantify nucleic acid analytes |
US20030165859A1 (en) * | 2001-10-23 | 2003-09-04 | Invitrogen Corporation | Primers and methods for the detection and discrimination of nucleic acids |
ATE328119T1 (en) * | 2001-11-02 | 2006-06-15 | Roche Diagnostics Gmbh | DETECTION OF VARIOLA VIRUS |
DE10153829A1 (en) * | 2001-11-05 | 2003-05-28 | Bayer Ag | Assay based on doped nanoparticles |
JP2005517900A (en) | 2001-11-21 | 2005-06-16 | アプレラ コーポレイション | Digital assay |
US7052877B2 (en) | 2001-11-30 | 2006-05-30 | Applera Corporation | Thermus brockianus nucleic acid polymerases |
WO2003048295A1 (en) | 2001-11-30 | 2003-06-12 | Fluidigm Corporation | Microfluidic device and methods of using same |
AU2002353043B2 (en) | 2001-11-30 | 2008-07-03 | The Johns Hopkins University | Methods for analyzing methylated CpG islands and GC rich regions |
US6686162B2 (en) | 2001-12-04 | 2004-02-03 | Quest Diagnostics Investments, Incorporated | Oligonucleotides and methods for detecting Borrelia burgdorferi |
US6635428B2 (en) | 2001-12-04 | 2003-10-21 | Quest Diagnostics Investments Incorporated | Oligonucleotides and methods for detecting hepatitis B viral nucleic acids |
US6946245B2 (en) | 2001-12-04 | 2005-09-20 | Quest Diagnostics Investments Incorporated | Oligonucleotides and methods for detecting hepatitis C viral nucleic acids |
US7198897B2 (en) * | 2001-12-19 | 2007-04-03 | Brandeis University | Late-PCR |
AU2002366803A1 (en) | 2001-12-19 | 2003-07-09 | Millennium Pharmaceuticals, Inc. | Human diacylglycerol acyltransferase 2 (dgat2) family members and uses therefor |
EP1953244A1 (en) | 2002-01-09 | 2008-08-06 | Nakamura, Yusuke | Cancer profiles |
US7255986B2 (en) * | 2002-01-31 | 2007-08-14 | The Board Of Trustees Operating Michigan State University | Compositions for the diagnosis and treatment of epizootic catarrhal enteritis in ferrets |
US20040166492A1 (en) * | 2002-02-05 | 2004-08-26 | Engel Stacia R. | Quantitative detection of dekkera and saccharomyces |
JP2006505832A (en) * | 2002-02-26 | 2006-02-16 | フアルマシア・コーポレーシヨン | Sequencing system calculator |
US6818420B2 (en) * | 2002-02-27 | 2004-11-16 | Biosource International, Inc. | Methods of using FET labeled oligonucleotides that include a 3′-5′ exonuclease resistant quencher domain and compositions for practicing the same |
EP1485056B1 (en) * | 2002-02-27 | 2011-04-06 | Life Technologies Corporation | Methods of using fet labeled oligonucleotides that include a'5' exonuclease resistant quencher domain and compositions for practicing the same |
EP1481092A4 (en) | 2002-03-01 | 2006-08-09 | Ravgen Inc | Methods for detection of genetic disorders |
US6977162B2 (en) * | 2002-03-01 | 2005-12-20 | Ravgen, Inc. | Rapid analysis of variations in a genome |
US7005267B2 (en) * | 2002-03-04 | 2006-02-28 | The United States Of America As Represented By The Secretary Of The Army | Internal positive control for probe-based nucleic acid molecule assays and methods of making and using thereof |
US7052848B2 (en) * | 2002-03-04 | 2006-05-30 | The United States Of America As Represented By The Secretary Of The Army | Internal positive control for probe-based nucleic acid molecule assays and methods of making and using thereof |
DE60207979T2 (en) * | 2002-03-05 | 2006-09-28 | Epigenomics Ag | Method and device for determining tissue specificity of free DNA in body fluids |
GB0205455D0 (en) * | 2002-03-07 | 2002-04-24 | Molecular Sensing Plc | Nucleic acid probes, their synthesis and use |
US20040115688A1 (en) * | 2002-04-19 | 2004-06-17 | Cheung Irene Y. | Detection of gd2 synthase mrna and uses thereof |
WO2003089670A2 (en) * | 2002-04-22 | 2003-10-30 | Lawler Joseph F Jr | Reagents for monitoring nuclei acid amplification and methods of using same |
US7442506B2 (en) * | 2002-05-08 | 2008-10-28 | Ravgen, Inc. | Methods for detection of genetic disorders |
US20070178478A1 (en) * | 2002-05-08 | 2007-08-02 | Dhallan Ravinder S | Methods for detection of genetic disorders |
US7727720B2 (en) * | 2002-05-08 | 2010-06-01 | Ravgen, Inc. | Methods for detection of genetic disorders |
US20030215814A1 (en) * | 2002-05-17 | 2003-11-20 | Cockerill Franklin R. | Detection of Shiga toxin- or Shiga-like toxin-producing organisms |
EP1546380A4 (en) * | 2002-05-28 | 2007-02-14 | Us Genomics Inc | Methods and apparati using single polymer analysis |
KR20050010902A (en) * | 2002-06-12 | 2005-01-28 | 카이론 코포레이션 | Identification of oligonucleotides for the capture, detection and ouantitation of hepatitis a viral nucleic acid |
AU2003244135A1 (en) * | 2002-06-21 | 2004-01-06 | Hinode Co, . Ltd. | Device for removing raindrops on umrella and system for promoting installation of the device |
DE60335116D1 (en) * | 2002-06-28 | 2011-01-05 | Primeradx Inc | METHOD FOR DETECTING SEQUENCE DIFFERENCES |
AU2003254298A1 (en) | 2002-08-02 | 2004-02-23 | Stratatech Corporation | Species specific dna detection |
EP2311978A1 (en) | 2002-08-20 | 2011-04-20 | Millennium Pharmaceuticals, Inc. | Compositions, kits, and methods for identification, assessment, prevention, and therapy of cervical cancer |
US8277753B2 (en) | 2002-08-23 | 2012-10-02 | Life Technologies Corporation | Microfluidic transfer pin |
CA2498320A1 (en) * | 2002-09-20 | 2004-04-01 | Integrated Dna Technologies, Inc. | Anthraquinone quencher dyes, their methods of preparation and use |
US7074598B2 (en) * | 2002-09-25 | 2006-07-11 | Mayo Foundation For Medical Education And Research | Detection of vancomycin-resistant enterococcus spp. |
US20070184453A1 (en) * | 2002-10-02 | 2007-08-09 | Roche Molecular Systems, Inc | Fret process |
US20040067492A1 (en) * | 2002-10-04 | 2004-04-08 | Allan Peng | Reverse transcription on microarrays |
US7384743B2 (en) * | 2002-10-16 | 2008-06-10 | University Of Miami | BRCA1/BCRA2 screening panel |
US6863731B2 (en) * | 2002-10-18 | 2005-03-08 | Controls Corporation Of America | System for deposition of inert barrier coating to increase corrosion resistance |
AU2003294716A1 (en) * | 2002-11-22 | 2004-06-18 | F.Hoffmann-La Roche Ag | Detectable labeled nucleoside analogs and methods of use thereof |
EP2112229A3 (en) | 2002-11-25 | 2009-12-02 | Sequenom, Inc. | Methods for identifying risk of breast cancer and treatments thereof |
WO2004051218A2 (en) * | 2002-12-04 | 2004-06-17 | Applera Corporation | Multiplex amplification of polynucleotides |
US20040156854A1 (en) | 2002-12-06 | 2004-08-12 | Millennium Pharmaceuticals, Inc. | Methods for the identification, assessment, and treatment of patients with proteasome inhibition therapy |
CN1506471A (en) * | 2002-12-09 | 2004-06-23 | Nuclein detecting method | |
US7291459B2 (en) * | 2002-12-10 | 2007-11-06 | University Of Alabama At Huntsville | Nucleic acid detector and method of detecting targets within a sample |
MXPA05006158A (en) * | 2002-12-12 | 2005-08-26 | Chiron Corp | Identification of oligonucleotides for the capture, detection and quantitation of west nile virus. |
US8206904B2 (en) | 2002-12-18 | 2012-06-26 | Third Wave Technologies, Inc. | Detection of nucleic acids |
CA2510381C (en) | 2002-12-18 | 2014-07-08 | Third Wave Technologies, Inc. | Detection of small nucleic acids |
EP2474630B1 (en) | 2002-12-20 | 2016-04-27 | Celera Corporation | Genetic polymorphisms associated with myocardial infarction, methods of detection and uses thereof |
CA2511381A1 (en) * | 2002-12-20 | 2004-07-22 | Stratagene California | Compositions and methods for polynucleotide detection |
AU2003302264A1 (en) | 2002-12-20 | 2004-09-09 | Biotrove, Inc. | Assay apparatus and method using microfluidic arrays |
DE10261468A1 (en) * | 2002-12-31 | 2004-07-15 | Bundesrepublik Deutschland, vertreten durch das Bundesministerium der Verteidigung, dieses vertreten durch das Bundesamt für Wehrtechnik und Beschaffung | Test kit for detecting Brucella species, potential biological warfare agents, in the environment, comprises primers for specific amplification, optionally also hybridization probe |
US7297780B2 (en) | 2003-01-06 | 2007-11-20 | Third Wave Technologies, Inc. | Reactive functional groups for preparation of modified nucleic acid |
CA2514877A1 (en) * | 2003-01-30 | 2004-08-12 | Bellbrook Labs, Llc | Assay method for group transfer reactions |
US8088897B2 (en) * | 2003-01-30 | 2012-01-03 | BellBrook Labs, Inc. | Assay method for group transfer reactions |
US7355010B2 (en) | 2003-01-30 | 2008-04-08 | Bellbrook Labs, Llc | Assay method for group transfer reactions |
EP1601955B1 (en) * | 2003-03-07 | 2013-01-09 | Luxcel Biosciences Limited | An oxygen sensitive probe and method for measuring oxygen uptake |
AU2004230569B2 (en) | 2003-03-31 | 2007-12-06 | F. Hoffmann-La Roche Ag | Compositions and methods for detecting certain flaviviruses, including members of the Japanese encephalitis virus serogroup |
WO2006102264A1 (en) | 2005-03-18 | 2006-09-28 | Fluidigm Corporation | Thermal reaction device and method for using the same |
JP5419248B2 (en) | 2003-04-03 | 2014-02-19 | フルイディグム コーポレイション | Microfluidic device and method of use thereof |
WO2004087950A2 (en) | 2003-04-04 | 2004-10-14 | Roche Diagnostics Gmbh | Improved system for multi color real time pcr |
US20040241732A1 (en) * | 2003-04-11 | 2004-12-02 | Chu-An Chang | Method of generating long nucleic acid molecules of defined sequence |
WO2004092327A2 (en) * | 2003-04-11 | 2004-10-28 | Applera Corporation | Method of generating size standard nucleic acids |
WO2004094986A2 (en) * | 2003-04-16 | 2004-11-04 | Handylab, Inc. | System and method for electrochemical detection of biological compounds |
CN1537954A (en) * | 2003-04-18 | 2004-10-20 | ������������ʽ���� | Expressing genetic analysis method and probe reagent box for expressing genetic analysng |
US20040214176A1 (en) * | 2003-04-22 | 2004-10-28 | Osborne James C. | Multiplexed DNA assays using structure-specific endonucleases |
US7892745B2 (en) * | 2003-04-24 | 2011-02-22 | Xdx, Inc. | Methods and compositions for diagnosing and monitoring transplant rejection |
US8679789B2 (en) * | 2003-05-01 | 2014-03-25 | Gen-Probe Incorporated | Oligonucleotides comprising a molecular switch |
JP4836795B2 (en) * | 2003-05-19 | 2011-12-14 | ブランデイズ ユニバーシティー | Nucleic acid processing method, kit, and apparatus |
PT1626714E (en) | 2003-05-20 | 2007-08-24 | Bayer Pharmaceuticals Corp | Diaryl ureas for diseases mediated by pdgfr |
EP2481814A3 (en) | 2003-06-09 | 2012-10-10 | The Regents of the University of Michigan | Compositions and methods for treating and diagnosing cancer |
WO2005003373A2 (en) * | 2003-06-26 | 2005-01-13 | Proligo, Llc | Fluorogenic nucleic acid probes including lna for methods to detect and/or quantify nucleic acid analytes |
US20050233314A1 (en) * | 2003-06-30 | 2005-10-20 | National Health Research Institutes | Sensitive and quantitative detection of pathogens by real-time nested PCR |
EP1502961B1 (en) * | 2003-08-01 | 2010-09-08 | Roche Diagnostics GmbH | New detection format for hot start real time polymerase chain reaction |
EP1502958A1 (en) * | 2003-08-01 | 2005-02-02 | Roche Diagnostics GmbH | New detection format for hot start real time polymerase chain reaction |
US7417726B2 (en) * | 2003-09-19 | 2008-08-26 | Applied Biosystems Inc. | Normalization of data using controls |
US20060029948A1 (en) * | 2003-09-19 | 2006-02-09 | Gary Lim | Sealing cover and dye compatibility selection |
US20050112634A1 (en) * | 2003-09-19 | 2005-05-26 | Woudenberg Timothy M. | High density sequence detection methods and apparatus |
MXPA06003402A (en) | 2003-10-07 | 2006-06-27 | Millennium Pharm Inc | Nucleic acid molecules and proteins for the identification, assessment, prevention, and therapy of ovarian cancer. |
US20050186588A1 (en) * | 2003-10-16 | 2005-08-25 | Third Wave Technologies, Inc. | Direct nucleic acid detection in bodily fluids |
CA2543830A1 (en) * | 2003-10-27 | 2005-05-19 | Monogram Biosciences, Inc. | Detecting human anti-therapeutic antibodies |
WO2005045061A2 (en) * | 2003-10-27 | 2005-05-19 | Hybridon, Inc. | Hybridization-based fluorescence assay |
SI1687609T1 (en) * | 2003-10-28 | 2015-03-31 | Epoch Biosciences, Inc. | Fluorescent probes for dna detection by hybridization with improved sensitivity and low background |
JP2007528213A (en) * | 2003-11-06 | 2007-10-11 | ユニバーシティ・オブ・ネバダ・リノ | Improved method for detection and measurement of specific nucleic acid sequences |
US7439341B2 (en) | 2003-11-14 | 2008-10-21 | Integrated Dna Technologies, Inc. | Fluorescence quenching azo dyes, their methods of preparation and use |
US7427475B2 (en) * | 2003-11-18 | 2008-09-23 | Mayo Foundation For Medical Education And Research | Detection of group B streptococcus |
US7667024B2 (en) * | 2003-11-19 | 2010-02-23 | Allelogic Biosciences Corp. | Oligonucleotides labeled with a plurality of fluorophores |
CA2991249C (en) | 2003-11-26 | 2020-07-07 | Celera Corporation | Single nucleotide polymorphisms associated with cardiovascular disorders and statin response, methods of detection and uses thereof |
JP2007512838A (en) | 2003-12-01 | 2007-05-24 | インヴィトロジェン コーポレーション | Nucleic acid molecules containing recombination sites and methods of use thereof |
CA2494571C (en) * | 2003-12-02 | 2010-02-09 | F.Hoffmann-La Roche Ag | Oligonucleotides containing molecular rods |
ATE557100T1 (en) | 2003-12-03 | 2012-05-15 | Abbott Lab | DOUBLE STRANDED LINEAR NUCLEIC ACID PROBE AND USE THEREOF |
EP1706836B1 (en) * | 2003-12-06 | 2011-01-19 | Abbott Laboratories | Method and system for analyzing reactions using an information system |
US8697433B2 (en) * | 2003-12-10 | 2014-04-15 | Samsung Electronics Co., Ltd. | Polymerase chain reaction (PCR) module and multiple PCR system using the same |
US20050147976A1 (en) * | 2003-12-29 | 2005-07-07 | Xing Su | Methods for determining nucleotide sequence information |
US7977048B2 (en) | 2004-01-13 | 2011-07-12 | Pathogenetix, Inc. | Detection and quantification of analytes in solution using polymers |
US7432057B2 (en) * | 2004-01-30 | 2008-10-07 | Michigan State University | Genetic test for PSE-susceptible turkeys |
CA2554779A1 (en) * | 2004-02-03 | 2005-08-18 | The Regents Of The University Of Michigan | Compositions and methods for characterizing, regulating, diagnosing, and treating cancer |
US7344701B2 (en) * | 2004-02-03 | 2008-03-18 | Biosearch Technologies, Inc. | Xanthene dyes |
US20050186606A1 (en) * | 2004-02-11 | 2005-08-25 | Schroeder Benjamin G. | Methods and compositions for detecting nucleic acids |
WO2005080596A1 (en) * | 2004-02-19 | 2005-09-01 | University College Cork - National University Of Ireland, Cork | Detection of biologically active compounds |
CA2557485A1 (en) * | 2004-02-26 | 2005-09-09 | Thomsen Bioscience A/S | Method, chip, device and integrated system for detection biological particles |
ATE474223T1 (en) * | 2004-02-26 | 2010-07-15 | Thomsen Bioscience As | METHOD, CHIP AND SYSTEM FOR COLLECTING BIOLOGICAL PARTICLES |
EP1730276B8 (en) * | 2004-02-26 | 2010-07-21 | DELTA Dansk Elektronik, Lys og Akustik | Method, chip, device and system for extraction of biological materials |
WO2005085475A1 (en) | 2004-03-01 | 2005-09-15 | Applera Corporation | Methods, compositions and kits for use in polynucleotide amplification |
EP1735097B1 (en) | 2004-03-12 | 2016-11-30 | Life Technologies Corporation | Nanoliter array loading |
GB0406015D0 (en) * | 2004-03-17 | 2004-04-21 | Dynal Biotech Asa | Improvements in magnetic polymer particles |
KR100906749B1 (en) * | 2004-03-25 | 2009-07-09 | (주)바이오니아 | Methods for Detecting Nucleic Acid Amplification Using Probe Labeled with Intercalating Dye |
US7445898B2 (en) * | 2004-04-01 | 2008-11-04 | Bio-Rad Laboratories, Inc. | Quantitative amplification with a labeled probe and 3′ to 5′ exonuclease activity |
ES2527528T3 (en) * | 2004-04-08 | 2015-01-26 | Sangamo Biosciences, Inc. | Methods and compositions to modulate cardiac contractility |
US7462451B2 (en) * | 2004-04-26 | 2008-12-09 | Third Wave Technologies, Inc. | Compositions for modifying nucleic acids |
CN101023170B (en) | 2004-04-26 | 2011-03-02 | 和光纯药工业株式会社 | Probe and primer for tubercle bacillus detection, and method of detecting human tubercle bacillus therewith |
EP2423327B1 (en) | 2004-05-07 | 2014-01-08 | Celera Corporation | Genetic polymorphism associated with liver fibrosis methods of detection and uses thereof |
US20080268476A1 (en) * | 2004-05-12 | 2008-10-30 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Nectin 4 (N4) as a Marker for Cancer Prognosis |
US20050255485A1 (en) * | 2004-05-14 | 2005-11-17 | Livak Kenneth J | Detection of gene duplications |
WO2005113804A1 (en) * | 2004-05-20 | 2005-12-01 | Trillion Genomics Limited | Use of mass labelled probes to detect target nucleic acids using mass spectrometry |
EP1766021B1 (en) * | 2004-05-20 | 2012-05-09 | AES Chemunex S.A. | Polynucleotides for the detection of escherichia coli o157:h7 and escherichia coli o157:nm verotoxin producers |
CA2572450A1 (en) * | 2004-05-28 | 2005-12-15 | Ambion, Inc. | Methods and compositions involving microrna |
US7575863B2 (en) * | 2004-05-28 | 2009-08-18 | Applied Biosystems, Llc | Methods, compositions, and kits comprising linker probes for quantifying polynucleotides |
US7279281B2 (en) * | 2004-06-01 | 2007-10-09 | The Regents Of The University Of Michigan | Methods and kits for diagnosing or monitoring autoimmune and chronic inflammatory diseases |
US20080152700A1 (en) | 2004-06-01 | 2008-06-26 | Reza Sheikhnejad | Methods and compositions for the inhibition of gene expression |
US7338763B2 (en) * | 2004-06-02 | 2008-03-04 | Eppendorf Array Technologies S.A. | Method and kit for the detection and/or quantification of homologous nucleotide sequences on arrays |
EP2701194B1 (en) | 2004-06-07 | 2018-08-29 | Fluidigm Corporation | Method and apparatus for imaging a microfluidic device under temperature control |
US20060003337A1 (en) * | 2004-06-30 | 2006-01-05 | John Brandis | Detection of small RNAS |
JP5020818B2 (en) | 2004-07-01 | 2012-09-05 | ジェン−プローブ・インコーポレーテッド | Methods and compositions for detecting nucleic acids in biological samples |
US20130055466A1 (en) | 2011-08-31 | 2013-02-28 | Monsanto Technology, Llc | Methods and Compositions for Watermelon Firmness |
AU2005263334C1 (en) * | 2004-07-20 | 2011-01-20 | Symphogen A/S | A procedure for structural characterization of a recombinant polyclonal protein or a polyclonal cell line |
MX2007000644A (en) * | 2004-07-20 | 2007-03-28 | Symphogen As | Anti-rhesus d recombinant polyclonal antibody and methods of manufacture. |
WO2006020933A2 (en) * | 2004-08-13 | 2006-02-23 | Stratagene California | Dual labeled fluorescent probes |
JP2008512099A (en) | 2004-09-07 | 2008-04-24 | テレソン インスティテュート フォー チャイルド ヘルス リサーチ | Substances for the treatment or prevention of allergic diseases |
WO2006029184A2 (en) * | 2004-09-08 | 2006-03-16 | Expression Diagnostics, Inc. | Genes useful for diagnosing and monitoring inflammation related disorders |
US20060051796A1 (en) * | 2004-09-09 | 2006-03-09 | Inga Boell | Real time PCR with the addition of pyrophosphatase |
EP1634965B1 (en) | 2004-09-09 | 2010-01-20 | Roche Diagnostics GmbH | Real time PCR with the addition of pyrophosphatase |
US7910753B2 (en) | 2004-09-10 | 2011-03-22 | Anaspec Incorporated | Cyanine dyes and their applications as luminescence quenching compounds |
JP2008513028A (en) * | 2004-09-21 | 2008-05-01 | アプレラ コーポレイション | Two-color real-time / endpoint quantification of microRNA (miRNA) |
US7851152B2 (en) * | 2004-09-25 | 2010-12-14 | Yaodong Chen | Fluorescent base analogues' usage in the characterization of nucleic acid molecules and their interactions |
US20060078894A1 (en) * | 2004-10-12 | 2006-04-13 | Winkler Matthew M | Methods and compositions for analyzing nucleic acids |
ES2668467T3 (en) | 2004-10-18 | 2018-05-18 | Brandeis University | LATE amplification methods and nucleic acid sequencing |
US20060147954A1 (en) * | 2004-10-19 | 2006-07-06 | Wallac Oy | Novel probe and its use in bioaffinity assays |
US20060115838A1 (en) * | 2004-10-19 | 2006-06-01 | Trevigen Inc. | Real-time detection of amplicons using nucleic acid repair enzymes |
WO2006045009A2 (en) * | 2004-10-20 | 2006-04-27 | Stratagene California | Triplex probe compositions and methods for polynucleotide detection |
ES2391744T3 (en) | 2004-11-01 | 2012-11-29 | George Mason University | Compositions and procedures for diagnosing colon disorders |
MX2007005364A (en) * | 2004-11-03 | 2008-01-22 | Third Wave Tech Inc | Single step detection assay. |
EP2302055B1 (en) | 2004-11-12 | 2014-08-27 | Asuragen, Inc. | Methods and compositions involving miRNA and miRNA inhibitor molecules |
US20060105348A1 (en) * | 2004-11-15 | 2006-05-18 | Lee Jun E | Compositions and methods for the detection and discrimination of nucleic acids |
US20060275792A1 (en) * | 2004-11-15 | 2006-12-07 | Lee Jun E | Enhancement of nucleic acid amplification using double-stranded DNA binding proteins |
KR20070090233A (en) * | 2004-12-08 | 2007-09-05 | 타케시 야마모토 | Method of examining gene sequence |
EP2208796B1 (en) | 2005-01-06 | 2014-06-18 | Applied Biosystems, LLC | Polypeptides having nucleic acid binding activity and compositions and methods for nucleic acid amplification |
US7315376B2 (en) * | 2005-01-07 | 2008-01-01 | Advanced Molecular Systems, Llc | Fluorescence detection system |
DE602005000877T2 (en) * | 2005-01-18 | 2007-12-20 | Roche Diagnostics Gmbh | Fluorescence imaging by telecentricity |
ES2284087T3 (en) * | 2005-01-18 | 2007-11-01 | F. Hoffmann-La Roche Ag | GENERATION OF FLUORESCENCE SIGNAL IMAGES BY TELECENTRIC EXCITATION AND IMAGE GENERATION OPTICS. |
US20070020644A1 (en) * | 2005-01-26 | 2007-01-25 | Kolykhalov Alexander A | Method for detection and characterization of short nucleic acids |
JP2008529528A (en) * | 2005-02-09 | 2008-08-07 | ストラタジーン カリフォルニア | Key probe composition and method for detection of polynucleotides |
JP2008531052A (en) * | 2005-02-28 | 2008-08-14 | ジェン−プローブ・インコーポレーテッド | Composition and method for detecting an analyte using a nucleic acid hybridization switch probe |
EP2314614B1 (en) * | 2005-02-28 | 2015-11-25 | Sangamo BioSciences, Inc. | Anti-angiogenic methods and compositions |
JP4398886B2 (en) * | 2005-03-07 | 2010-01-13 | ソニー株式会社 | COMMUNICATION TERMINAL DEVICE, COMMUNICATION SYSTEM, COMMUNICATION METHOD, AND PROGRAM |
US8263330B1 (en) | 2005-03-08 | 2012-09-11 | Quest Diagnostics Investments Incorporated | Detection of Mycobacterium tuberculosis complex nucleic acids |
US7759469B2 (en) | 2005-03-10 | 2010-07-20 | Roche Diagnostics Operations, Inc. | Labeling reagent |
US7964413B2 (en) | 2005-03-10 | 2011-06-21 | Gen-Probe Incorporated | Method for continuous mode processing of multiple reaction receptacles in a real-time amplification assay |
EP1700922B1 (en) | 2005-03-10 | 2016-08-24 | Roche Diagnostics GmbH | 3-Substituted 5-Nitroindole derivatives and labeled oligonucleotide probes containing them |
JP5590697B2 (en) | 2005-03-11 | 2014-09-17 | セレラ コーポレーション | Genetic polymorphism associated with coronary heart disease, detection method and use thereof |
US7776567B2 (en) * | 2005-03-17 | 2010-08-17 | Biotium, Inc. | Dimeric and trimeric nucleic acid dyes, and associated systems and methods |
US7601498B2 (en) * | 2005-03-17 | 2009-10-13 | Biotium, Inc. | Methods of using dyes in association with nucleic acid staining or detection and associated technology |
US20060246493A1 (en) | 2005-04-04 | 2006-11-02 | Caliper Life Sciences, Inc. | Method and apparatus for use in temperature controlled processing of microfluidic samples |
US20060275798A1 (en) * | 2005-04-04 | 2006-12-07 | Steichen John C | Detection of organisms using a media sachet and primer directed nucleic acid amplification |
US20070213293A1 (en) * | 2005-04-08 | 2007-09-13 | Nastech Pharmaceutical Company Inc. | Rnai therapeutic for respiratory virus infection |
KR20070118703A (en) * | 2005-04-08 | 2007-12-17 | 나스텍 파마수티컬 컴퍼니 인코포레이티드 | Rnai therapeutic for respiratory virus infection |
EP1877578A4 (en) * | 2005-04-28 | 2008-06-25 | Merck & Co Inc | Fluorescent multiplex hpv pcr assays |
EP1877585A2 (en) * | 2005-04-28 | 2008-01-16 | Merck & Co., Inc. | Real-time hpv pcr assays |
CA2607369A1 (en) * | 2005-05-02 | 2006-11-09 | Stratagene California | Oligonucleotide probe/primer compositions and methods for polynucleotide detection |
EP1886145A2 (en) | 2005-05-03 | 2008-02-13 | Applera Corporation | Fluorescent detection system and dye set for use therewith |
WO2006124664A2 (en) * | 2005-05-12 | 2006-11-23 | Third Wave Technologies, Inc. | Polymorphic ghsr nucleic acids and uses thereof |
CN102539734B (en) | 2005-05-12 | 2016-02-03 | 清华大学 | The cancer diagnosis that paranuclein is auxiliary and methods for the treatment of |
CN103103181B (en) | 2005-05-13 | 2015-01-21 | 和光纯药工业株式会社 | Primers, probes, methods and uses thereof for the detection of mycobacterium kansasii |
WO2006127507A2 (en) | 2005-05-20 | 2006-11-30 | Integrated Dna Technologies, Inc. | Compounds and methods for labeling oligonucleotides |
US7737281B2 (en) * | 2005-05-24 | 2010-06-15 | Enzo Life Sciences, Inc. C/O Enzo Biochem, Inc. | Purine based fluorescent dyes |
US8362250B2 (en) | 2005-05-24 | 2013-01-29 | Enzo Biochem, Inc. | Fluorescent dyes and compounds, methods and kits useful for identifying specific organelles and regions in cells of interest |
US8357801B2 (en) | 2005-05-24 | 2013-01-22 | Enzo Life Sciences, Inc. | Labeling of target molecules, identification of organelles and other applications, novel compositions, methods and kits |
US7569695B2 (en) * | 2005-05-24 | 2009-08-04 | Enzo Life Sciences, Inc. | Dyes for the detection or quantification of desirable target molecules |
GB0510979D0 (en) | 2005-05-28 | 2005-07-06 | Kbiosciences Ltd | Detection system for PCR assay |
US7439024B2 (en) | 2005-06-01 | 2008-10-21 | The United States Of America As Represented By The Department Of Veterans Affairs | Methods and kits for diagnosing or monitoring autoimmune and chronic inflammatory diseases |
US20090317798A1 (en) * | 2005-06-02 | 2009-12-24 | Heid Christian A | Analysis using microfluidic partitioning devices |
US20070099209A1 (en) * | 2005-06-13 | 2007-05-03 | The Regents Of The University Of Michigan | Compositions and methods for treating and diagnosing cancer |
JP2008546387A (en) * | 2005-06-13 | 2008-12-25 | ザ リージェンツ オブ ザ ユニバーシティ オブ ミシガン | Compositions and methods for treating and diagnosing cancer |
JP2008543300A (en) | 2005-06-16 | 2008-12-04 | バイオツールズ バイオテクノロジカル アンド メディカル ラボラトリーズ, エス. アー. | Nucleic acid detection method using direct generation of measurable signal |
EP1907592B1 (en) * | 2005-07-01 | 2011-03-09 | Dako Denmark A/S | Monomeric and polymeric linkers useful for conjugating biological molecules and other substances |
US7977108B2 (en) * | 2005-07-25 | 2011-07-12 | Roche Molecular Systems, Inc. | Method for detecting a mutation in a repetitive nucleic acid sequence |
AU2006272568B2 (en) * | 2005-07-26 | 2012-06-28 | Merck Sharp & Dohme Corp. | Assays for resistance to echinocandin-class drugs |
US7892795B2 (en) * | 2005-08-02 | 2011-02-22 | Focus Diagnostics, Inc. | Methods and compositions for detecting BK virus |
WO2007019410A2 (en) * | 2005-08-05 | 2007-02-15 | The Board Of Trustees Of The Leland Stanford Junior University | Mammalian obestatin receptors |
ES2494922T3 (en) * | 2005-09-01 | 2014-09-16 | Ausdiagnostics Pty Ltd. | Methods for amplification, quantification and identification of nucleic acids |
US20070059713A1 (en) * | 2005-09-09 | 2007-03-15 | Lee Jun E | SSB-DNA polymerase fusion proteins |
US7799530B2 (en) | 2005-09-23 | 2010-09-21 | Celera Corporation | Genetic polymorphisms associated with cardiovascular disorders and drug response, methods of detection and uses thereof |
US8831887B2 (en) * | 2005-10-12 | 2014-09-09 | The Research Foundation For The State University Of New York | Absolute PCR quantification |
EP1777298A1 (en) * | 2005-10-13 | 2007-04-25 | Roche Diagnostics GmbH | Multiplexed solid-phase nucleic acid amplification assay |
US8652467B2 (en) * | 2005-10-14 | 2014-02-18 | The Regents Of The University Of Michigan | Dek protein compositions and methods of using the same |
US7781165B2 (en) | 2005-10-19 | 2010-08-24 | Roche Diagnostics Operations, Inc. | Benzimidazolium compounds and salts of benzimidazolium compounds for nucleic acid amplification |
WO2007045890A1 (en) * | 2005-10-21 | 2007-04-26 | Primerdesign Ltd | Probe |
US7723477B2 (en) | 2005-10-31 | 2010-05-25 | Oncomed Pharmaceuticals, Inc. | Compositions and methods for inhibiting Wnt-dependent solid tumor cell growth |
CA2628255C (en) * | 2005-10-31 | 2016-04-19 | The Regents Of The University Of Michigan | Compositions and methods for treating and diagnosing cancer |
ES2618785T3 (en) | 2005-10-31 | 2017-06-22 | Oncomed Pharmaceuticals, Inc. | Compositions and methods for treating cancer based on human FZD receptors |
US20100035247A1 (en) * | 2005-11-04 | 2010-02-11 | U.S. Genomics, Inc. | Heterogeneous Assay of Analytes in Solution Using Polymers |
ES2332139T3 (en) | 2005-11-23 | 2010-01-27 | F. Hoffmann-La Roche Ag | POLINUCLEOTIDOS WITH PHOSPHATE MIMETIC. |
US20090068643A1 (en) * | 2005-11-23 | 2009-03-12 | Integrated Dna Technologies, Inc. | Dual Function Primers for Amplifying DNA and Methods of Use |
US7824858B2 (en) * | 2006-01-23 | 2010-11-02 | Quest Diagnostics Investments Incorporated | Assay for mycobacterium avium/intracellulare nucleic acid |
EP1998785A4 (en) * | 2006-02-21 | 2009-06-17 | Univ Michigan | Hedgehog signaling pathway antagonist cancer treatment |
CN101421417B (en) * | 2006-02-27 | 2013-04-03 | 霍夫曼-拉罗奇有限公司 | PCR hot start by magnesium sequestration |
CA2638739A1 (en) | 2006-03-01 | 2007-09-13 | Pioneer Hi-Bred International, Inc. | Compositions related to the quantitative trait locus 6 (qtl6) in maize and methods of use |
WO2007105673A1 (en) | 2006-03-13 | 2007-09-20 | Wako Pure Chemical Industries, Ltd. | Method for detection of mutant gene |
RU2394915C2 (en) * | 2006-03-24 | 2010-07-20 | Александр Борисович Четверин | Non-contact methods of detecting molecular colonies, sets of reagents and device for realising said methods |
EP2007909A4 (en) * | 2006-04-07 | 2011-01-26 | Xdx Inc | Steroid responsive nucleic acid expression and prediction of disease activity |
US20070238095A1 (en) * | 2006-04-11 | 2007-10-11 | Mayo Foundation For Medical Education And Research , A Minnesota Corporation | Detection of Influenza A Virus |
US20070238093A1 (en) * | 2006-04-11 | 2007-10-11 | Espy Mark J | Detection of influenza A virus |
US8828661B2 (en) | 2006-04-24 | 2014-09-09 | Fluidigm Corporation | Methods for detection and quantification of nucleic acid or protein targets in a sample |
ES2700606T3 (en) | 2006-04-28 | 2019-02-18 | Igor Kutyavin | Use of modified deoxynucleoside triphosphates in their bases to improve the detection of nucleic acids |
EP2021506B1 (en) * | 2006-04-28 | 2016-03-16 | Igor Kutyavin | Use of products of pcr amplification carrying elements of secondary structure to improve pcr-based nucleic acid detection |
US8188256B2 (en) | 2006-05-02 | 2012-05-29 | Wako Pure Chemical Industries, Ltd. | Primer and probe for detection of Mycobacterium intracellulare |
US8232091B2 (en) * | 2006-05-17 | 2012-07-31 | California Institute Of Technology | Thermal cycling system |
CN101541975B (en) | 2006-06-01 | 2013-04-03 | 第三次浪潮技术公司 | Detection of nucleic acids |
US8153369B2 (en) | 2006-06-05 | 2012-04-10 | Cancer Care Ontario | Assessment of risk for colorectal cancer |
US11001881B2 (en) | 2006-08-24 | 2021-05-11 | California Institute Of Technology | Methods for detecting analytes |
US7759062B2 (en) * | 2006-06-09 | 2010-07-20 | Third Wave Technologies, Inc. | T-structure invasive cleavage assays, consistent nucleic acid dispensing, and low level target nucleic acid detection |
US7951995B2 (en) | 2006-06-28 | 2011-05-31 | Pioneer Hi-Bred International, Inc. | Soybean event 3560.4.3.5 and compositions and methods for the identification and detection thereof |
EP2442108B1 (en) | 2006-07-14 | 2016-11-16 | The Regents of The University of California | Cancer biomarkers and methods of use thereof |
CA2658071C (en) | 2006-07-17 | 2013-05-28 | Brandeis University | Specialized oligonucleotides and their use in nucleic acid amplification and detection |
US8048626B2 (en) | 2006-07-28 | 2011-11-01 | California Institute Of Technology | Multiplex Q-PCR arrays |
US11525156B2 (en) | 2006-07-28 | 2022-12-13 | California Institute Of Technology | Multiplex Q-PCR arrays |
EP2484781B1 (en) | 2006-08-01 | 2017-08-23 | Applied Biosystems, LLC | Detection of analytes and nucleic acids |
US7993832B2 (en) * | 2006-08-14 | 2011-08-09 | Xdx, Inc. | Methods and compositions for diagnosing and monitoring the status of transplant rejection and immune disorders |
JP5088034B2 (en) * | 2006-08-14 | 2012-12-05 | ソニー株式会社 | Nucleic acid strands useful for detecting substances and methods |
WO2008021446A2 (en) * | 2006-08-15 | 2008-02-21 | Genetag Technology, Inc. | Probe-antiprobe compositions and methods for dna or rna detection |
US11560588B2 (en) | 2006-08-24 | 2023-01-24 | California Institute Of Technology | Multiplex Q-PCR arrays |
US20080261216A1 (en) * | 2006-09-08 | 2008-10-23 | The Regents Of The University Of Michigan | HERV Group II Viruses In Lymphoma And Cancer |
US8055034B2 (en) | 2006-09-13 | 2011-11-08 | Fluidigm Corporation | Methods and systems for image processing of microfluidic devices |
US8050516B2 (en) * | 2006-09-13 | 2011-11-01 | Fluidigm Corporation | Methods and systems for determining a baseline during image processing |
EP2145001A2 (en) * | 2006-09-19 | 2010-01-20 | Asuragen, Inc. | Mir-15, mir-26, mir -31,mir -145, mir-147, mir-188, mir-215, mir-216 mir-331, mmu-mir-292-3p regulated genes and pathways as targets for therapeutic intervention |
AU2007307171B2 (en) * | 2006-10-04 | 2012-01-19 | Third Wave Technologies, Inc. | Snap-back primers and detectable hairpin structures |
US20100136537A1 (en) * | 2006-10-10 | 2010-06-03 | The Regents Of The University Of Michigan | Photoreceptor precursor cells |
CA2915679C (en) | 2006-10-20 | 2017-12-12 | Celera Corporation | Genetic polymorphisms associated with venous thrombosis, methods of detection and uses thereof |
WO2008054747A2 (en) | 2006-10-31 | 2008-05-08 | E. I. Du Pont De Nemours And Company | Soybean event dp-305423-1 and compositions and methods for the identification and/or detection thereof |
CA2668457C (en) * | 2006-11-02 | 2016-10-04 | Veridex, Llc | Imaging of activated vascular endothelium using immunomagnetic mri contrast agents |
WO2008058018A2 (en) | 2006-11-02 | 2008-05-15 | Mayo Foundation For Medical Education And Research | Predicting cancer outcome |
US8148067B2 (en) | 2006-11-09 | 2012-04-03 | Xdx, Inc. | Methods for diagnosing and monitoring the status of systemic lupus erythematosus |
WO2008067552A2 (en) * | 2006-11-30 | 2008-06-05 | Fluidigm Corporation | Method and apparatus for biological sample analysis |
US20080131878A1 (en) * | 2006-12-05 | 2008-06-05 | Asuragen, Inc. | Compositions and Methods for the Detection of Small RNA |
EP2104737B1 (en) * | 2006-12-08 | 2013-04-10 | Asuragen, INC. | Functions and targets of let-7 micro rnas |
CA2671194A1 (en) * | 2006-12-08 | 2008-06-19 | Asuragen, Inc. | Mir-20 regulated genes and pathways as targets for therapeutic intervention |
WO2008073915A2 (en) * | 2006-12-08 | 2008-06-19 | Asuragen, Inc. | Micrornas differentially expressed in leukemia and uses thereof |
CA2671294A1 (en) * | 2006-12-08 | 2008-06-19 | Asuragen, Inc. | Mir-21 regulated genes and pathways as targets for therapeutic intervention |
CN101657548B (en) | 2006-12-13 | 2012-10-10 | 卢米耐克斯公司 | Systems and methods for multiplex analysis of PCR in real time |
EP2383349B1 (en) | 2006-12-18 | 2014-12-31 | Wako Pure Chemical Industries, Ltd. | Primer and probe for detection of mycobacterium avium and method for detection of mycobacterium avium using the same |
US9938641B2 (en) * | 2006-12-18 | 2018-04-10 | Fluidigm Corporation | Selection of aptamers based on geometry |
JP2010514692A (en) | 2006-12-20 | 2010-05-06 | バイエル ヘルスケア リミティド ライアビリティ カンパニー | Hydroxymethylphenylpyrazolylurea compounds useful for the treatment of cancer |
AU2007339793A1 (en) * | 2006-12-29 | 2008-07-10 | Applied Biosystems, Llc | Systems and methods for detecting nucleic acids |
US20080241838A1 (en) * | 2006-12-29 | 2008-10-02 | Applera Corporation, Applied Biosystems Group | Methods and systems for detecting nucleic acids |
US20090175827A1 (en) * | 2006-12-29 | 2009-07-09 | Byrom Mike W | miR-16 REGULATED GENES AND PATHWAYS AS TARGETS FOR THERAPEUTIC INTERVENTION |
EP2106439B1 (en) | 2007-01-24 | 2014-11-12 | The Regents of the University of Michigan | Compositions and methods for treating and diagnosing pancreatic cancer |
JP5913785B2 (en) | 2007-01-31 | 2016-05-25 | セレラ コーポレーション | Molecular prognostic signature and its use to predict distant metastasis of breast cancer |
US20100129792A1 (en) * | 2007-02-06 | 2010-05-27 | Gerassimos Makrigiorgos | Direct monitoring and pcr amplification of the dosage and dosage difference between target genetic regions |
GB0703996D0 (en) * | 2007-03-01 | 2007-04-11 | Oxitec Ltd | Nucleic acid detection |
GB0703997D0 (en) * | 2007-03-01 | 2007-04-11 | Oxitec Ltd | Methods for detecting nucleic sequences |
CA2679954A1 (en) | 2007-03-05 | 2008-09-12 | Cancer Care Ontario | Assessment of risk for colorectal cancer |
ES2582602T3 (en) | 2007-03-28 | 2016-09-14 | Signal Diagnostics | High resolution nucleic acid analysis system and method to detect sequence variations |
JP5191041B2 (en) * | 2007-04-05 | 2013-04-24 | エフ.ホフマン−ラ ロシュ アーゲー | Rapid one-step RT-PCR |
US9290803B2 (en) * | 2007-04-12 | 2016-03-22 | University Of Southern California | DNA methylation analysis by digital bisulfite genomic sequencing and digital methylight |
US20080274458A1 (en) * | 2007-05-01 | 2008-11-06 | Latham Gary J | Nucleic acid quantitation methods |
EP2639315A1 (en) | 2007-05-11 | 2013-09-18 | The Johns Hopkins University | Biomarkers for melanoma |
US20090232893A1 (en) * | 2007-05-22 | 2009-09-17 | Bader Andreas G | miR-143 REGULATED GENES AND PATHWAYS AS TARGETS FOR THERAPEUTIC INTERVENTION |
WO2008151004A1 (en) | 2007-05-31 | 2008-12-11 | Yale University | A genetic lesion associated with cancer |
US9271455B2 (en) * | 2007-05-31 | 2016-03-01 | Monsanto Technology Llc | Soybean polymorphisms and methods of genotyping |
EP2167138A2 (en) * | 2007-06-08 | 2010-03-31 | Asuragen, INC. | Mir-34 regulated genes and pathways as targets for therapeutic intervention |
EP2016821A1 (en) | 2007-06-13 | 2009-01-21 | Syngeta Participations AG | New hybrid system for Brassica napus |
DE102007029772B4 (en) | 2007-06-22 | 2011-12-08 | Aj Innuscreen Gmbh | Method and rapid test for the detection of specific nucleic acid sequences |
US8008010B1 (en) * | 2007-06-27 | 2011-08-30 | Applied Biosystems, Llc | Chimeric oligonucleotides for ligation-enhanced nucleic acid detection, methods and compositions therefor |
US20090324596A1 (en) | 2008-06-30 | 2009-12-31 | The Trustees Of Princeton University | Methods of identifying and treating poor-prognosis cancers |
US10745701B2 (en) | 2007-06-28 | 2020-08-18 | The Trustees Of Princeton University | Methods of identifying and treating poor-prognosis cancers |
US8158758B2 (en) | 2007-07-02 | 2012-04-17 | Oncomed Pharmaceuticals, Inc. | Compositions and methods for treating and diagnosing cancer |
IL184478A (en) * | 2007-07-08 | 2017-07-31 | Hadasit Medical Res Services & Development Ltd | Compositions, methods and kits for the diagnosis of carriers of mutations in the brca1 and brca2 genes and early diagnosis of cancerous disorders associated with mutations in brca1 and brca2 genes |
EP2735619A3 (en) | 2007-08-29 | 2014-08-13 | Monsanto Technology LLC | Methods and compositions for breeding for preferred traits associated with Goss' Wilt resistance in plants |
EP2198293B1 (en) | 2007-09-07 | 2012-01-18 | Fluidigm Corporation | Copy number variation determination, methods and systems |
US20100173294A1 (en) * | 2007-09-11 | 2010-07-08 | Roche Molecular Systems, Inc. | Diagnostic test for susceptibility to b-raf kinase inhibitors |
US8361714B2 (en) * | 2007-09-14 | 2013-01-29 | Asuragen, Inc. | Micrornas differentially expressed in cervical cancer and uses thereof |
US20090186015A1 (en) * | 2007-10-18 | 2009-07-23 | Latham Gary J | Micrornas differentially expressed in lung diseases and uses thereof |
US8039212B2 (en) | 2007-11-05 | 2011-10-18 | Celera Corporation | Genetic polymorphisms associated with liver fibrosis, methods of detection and uses thereof |
WO2009064766A1 (en) | 2007-11-16 | 2009-05-22 | E. I. Du Pont De Nemours And Company | Method for detection and/or analysis of yeast and mold in filterable liquids |
WO2009070805A2 (en) | 2007-12-01 | 2009-06-04 | Asuragen, Inc. | Mir-124 regulated genes and pathways as targets for therapeutic intervention |
US20090192114A1 (en) * | 2007-12-21 | 2009-07-30 | Dmitriy Ovcharenko | miR-10 Regulated Genes and Pathways as Targets for Therapeutic Intervention |
WO2009091556A2 (en) * | 2008-01-17 | 2009-07-23 | The General Hospital Corporation | Diagnostic methods and kits using fibroblast growth factor-23 |
EP2238459B1 (en) * | 2008-01-23 | 2019-05-08 | Roche Diagnostics GmbH | Integrated instrument performing synthesis and amplification |
US20090203022A1 (en) * | 2008-02-07 | 2009-08-13 | Arizona Board Of Regents For And On Behalf Of Arizona State University | Analysis |
WO2009100430A2 (en) * | 2008-02-08 | 2009-08-13 | Asuragen, Inc | miRNAs DIFFERENTIALLY EXPRESSED IN LYMPH NODES FROM CANCER PATIENTS |
EP2251422B1 (en) | 2008-02-08 | 2013-04-10 | Wako Pure Chemical Industries, Ltd. | Primer and probe for detecting chlamydophilia caviae, as well as chlamydophilia caviae detection method using the same |
US9157116B2 (en) | 2008-02-08 | 2015-10-13 | Fluidigm Corporation | Combinatorial amplification and detection of nucleic acids |
JP5665548B2 (en) * | 2008-02-08 | 2015-02-04 | メイヨ・ファウンデーション・フォー・メディカル・エデュケーション・アンド・リサーチ | Clostridium difficile detection |
US20090221620A1 (en) | 2008-02-20 | 2009-09-03 | Celera Corporation | Gentic polymorphisms associated with stroke, methods of detection and uses thereof |
JP5651022B2 (en) * | 2008-03-15 | 2015-01-07 | ホロジック,インコーポレーテッド | Compositions and methods for analyzing nucleic acid molecules in amplification reactions |
WO2009154835A2 (en) * | 2008-03-26 | 2009-12-23 | Asuragen, Inc. | Compositions and methods related to mir-16 and therapy of prostate cancer |
PL2757091T3 (en) | 2008-04-01 | 2017-10-31 | Biosearch Tech Inc | Stabilized Nucleic Acid Dark Quencher-Fluorophore Probes |
US20090253121A1 (en) * | 2008-04-04 | 2009-10-08 | Micah Halpern | Method for amt-rflp dna fingerprinting |
WO2009126726A1 (en) * | 2008-04-08 | 2009-10-15 | Asuragen, Inc | Methods and compositions for diagnosing and modulating human papillomavirus (hpv) |
GB0806676D0 (en) | 2008-04-12 | 2008-05-14 | Environment Agency The | Environmental monitoring |
BRPI0911551B8 (en) | 2008-04-24 | 2023-10-03 | Monsanto Technology Llc | Method to produce soybean plant resistant to Asian soybean rust (ASR) |
EP2990487A1 (en) * | 2008-05-08 | 2016-03-02 | Asuragen, INC. | Compositions and methods related to mirna modulation of neovascularization or angiogenesis |
EP2288726B1 (en) | 2008-05-13 | 2012-06-13 | Gen-Probe Incorporated | Inactivatable target capture oligomers for use in the selective hybridization and capture of target nucleic acid sequences |
WO2009145181A1 (en) | 2008-05-28 | 2009-12-03 | 和光純薬工業株式会社 | Primer and probe for detection of mycobacterium intracellulare, and method for detection of mycobacterium intracellulare using the primer or the probe |
EP2291553A4 (en) | 2008-05-28 | 2011-12-14 | Genomedx Biosciences Inc | Systems and methods for expression-based discrimination of distinct clinical disease states in prostate cancer |
US10407731B2 (en) | 2008-05-30 | 2019-09-10 | Mayo Foundation For Medical Education And Research | Biomarker panels for predicting prostate cancer outcomes |
US20090305251A1 (en) * | 2008-06-09 | 2009-12-10 | University Of Miami | Brca1/brca2 screening panel |
EP2733222A1 (en) | 2008-07-09 | 2014-05-21 | Celera Corporation | Genetic polymorphisms associated with cardiovascular diseases, methods of detection and uses thereof |
US20100092960A1 (en) * | 2008-07-25 | 2010-04-15 | Pacific Biosciences Of California, Inc. | Helicase-assisted sequencing with molecular beacons |
EP2148187A1 (en) | 2008-07-25 | 2010-01-27 | Roche Diagnostics GmbH | Stimulation and optical display system for fluorescence detection |
CN102112631A (en) * | 2008-08-01 | 2011-06-29 | 霍夫曼-拉罗奇有限公司 | Improved lysis and reverse transcription for mRNA quantification |
US9182406B2 (en) * | 2008-08-04 | 2015-11-10 | Biodesy, Inc. | Nonlinear optical detection of molecules comprising an unnatural amino acid possessing a hyperpolarizability |
EP2321427B1 (en) | 2008-08-12 | 2017-11-29 | Stokes Bio Limited | Methods for digital pcr |
WO2010021936A1 (en) * | 2008-08-16 | 2010-02-25 | The Board Of Trustees Of The Leland Stanford Junior University | Digital pcr calibration for high throughput sequencing |
EP2326957B1 (en) * | 2008-09-03 | 2013-06-12 | Abbott Molecular Inc. | Assays and kits for determining hiv-1 tropism |
US9334281B2 (en) * | 2008-09-08 | 2016-05-10 | Enzo Life Sciences, Inc. | Fluorochromes for organelle tracing and multi-color imaging |
US9250249B2 (en) | 2008-09-08 | 2016-02-02 | Enzo Biochem, Inc. | Autophagy and phospholipidosis pathway assays |
CA2732750A1 (en) | 2008-09-22 | 2010-03-25 | The Government Of The United States Of America D.B.A.The Department Of V Eterans Affairs | Methods for detecting a mycobacterium tuberculosis infection |
US9399215B2 (en) | 2012-04-13 | 2016-07-26 | Bio-Rad Laboratories, Inc. | Sample holder with a well having a wicking promoter |
WO2011120024A1 (en) | 2010-03-25 | 2011-09-29 | Quantalife, Inc. | Droplet generation for droplet-based assays |
US9417190B2 (en) | 2008-09-23 | 2016-08-16 | Bio-Rad Laboratories, Inc. | Calibrations and controls for droplet-based assays |
US8709762B2 (en) | 2010-03-02 | 2014-04-29 | Bio-Rad Laboratories, Inc. | System for hot-start amplification via a multiple emulsion |
US9156010B2 (en) | 2008-09-23 | 2015-10-13 | Bio-Rad Laboratories, Inc. | Droplet-based assay system |
US9132394B2 (en) | 2008-09-23 | 2015-09-15 | Bio-Rad Laboratories, Inc. | System for detection of spaced droplets |
US10512910B2 (en) | 2008-09-23 | 2019-12-24 | Bio-Rad Laboratories, Inc. | Droplet-based analysis method |
US11130128B2 (en) | 2008-09-23 | 2021-09-28 | Bio-Rad Laboratories, Inc. | Detection method for a target nucleic acid |
US8633015B2 (en) | 2008-09-23 | 2014-01-21 | Bio-Rad Laboratories, Inc. | Flow-based thermocycling system with thermoelectric cooler |
US8951939B2 (en) | 2011-07-12 | 2015-02-10 | Bio-Rad Laboratories, Inc. | Digital assays with multiplexed detection of two or more targets in the same optical channel |
US9764322B2 (en) | 2008-09-23 | 2017-09-19 | Bio-Rad Laboratories, Inc. | System for generating droplets with pressure monitoring |
US9492797B2 (en) | 2008-09-23 | 2016-11-15 | Bio-Rad Laboratories, Inc. | System for detection of spaced droplets |
CN105079805A (en) | 2008-09-26 | 2015-11-25 | 昂考梅德药品有限公司 | Frizzled-binding agents and uses thereof |
CA2682439A1 (en) | 2008-10-17 | 2010-04-17 | F. Hoffmann-La Roche Ag | Cell monitoring and molecular analysis |
EP2687609B1 (en) | 2008-11-10 | 2017-01-04 | The United States of America, as represented by The Secretary, Department of Health and Human Services | Method for treating solid tumor |
RS55784B1 (en) | 2008-11-11 | 2017-07-31 | Univ Michigan Regents | Anti-cxcr1 compositions and methods |
US20100179213A1 (en) * | 2008-11-11 | 2010-07-15 | Mirna Therapeutics, Inc. | Methods and Compositions Involving miRNAs In Cancer Stem Cells |
GB2463401B (en) | 2008-11-12 | 2014-01-29 | Caris Life Sciences Luxembourg Holdings S A R L | Characterizing prostate disorders by analysis of microvesicles |
US10236078B2 (en) | 2008-11-17 | 2019-03-19 | Veracyte, Inc. | Methods for processing or analyzing a sample of thyroid tissue |
US9495515B1 (en) | 2009-12-09 | 2016-11-15 | Veracyte, Inc. | Algorithms for disease diagnostics |
US10669574B2 (en) | 2008-11-18 | 2020-06-02 | XCR Diagnostics, Inc. | DNA amplification technology |
CN102292457B (en) | 2008-11-25 | 2014-04-09 | 简·探针公司 | Compositions and methods for detecting small RNAS, and uses thereof |
US20100150949A1 (en) * | 2008-12-16 | 2010-06-17 | Oncopharmacologics, Inc. | Methods and compositions for modulating proline levels |
CN102301005A (en) | 2008-12-17 | 2011-12-28 | 生命技术公司 | Methods, compositions, and kits for detecting allelic variants |
US8362318B2 (en) | 2008-12-18 | 2013-01-29 | Board Of Trustees Of Michigan State University | Enzyme directed oil biosynthesis in microalgae |
EP2379739A1 (en) | 2008-12-19 | 2011-10-26 | Monsanto Invest N.V. | Method of breeding cysdv-resistant cucumber plants |
CA2688174C (en) * | 2008-12-19 | 2018-08-07 | F. Hoffmann-La Roche Ag | Dry composition of reaction compounds with stabilized polymerase |
US20100159452A1 (en) * | 2008-12-22 | 2010-06-24 | Roche Molecular Systems, Inc. | Method For Detecting a Target Nucleic Acid in a Sample |
US8628923B2 (en) | 2009-01-13 | 2014-01-14 | Fluidigm Corporation | Single cell nucleic acid analysis |
EP2381965B1 (en) | 2009-01-14 | 2020-05-06 | Drexel University | Modulation of pre-mrna using splice modulating oligonucleotides as therapeutic agents in the treatment of disease |
WO2010101696A1 (en) | 2009-02-06 | 2010-09-10 | Yale University | A snp marker of breast and ovarian cancer risk |
EP2393941A2 (en) * | 2009-02-09 | 2011-12-14 | Frederic Zenhausern | Improvements in and relating to microfluidic devices for processing a sample |
WO2010093872A2 (en) | 2009-02-13 | 2010-08-19 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Molecular-based method of cancer diagnosis and prognosis |
EP2399131B1 (en) | 2009-02-20 | 2014-08-27 | The U.S.A. As Represented By The Secretary, Department Of Health And Human Services | Method for the diagnosis of age-associated vascular disorders |
US9074258B2 (en) | 2009-03-04 | 2015-07-07 | Genomedx Biosciences Inc. | Compositions and methods for classifying thyroid nodule disease |
US8039215B2 (en) | 2009-03-10 | 2011-10-18 | Roche Molecular Systems, Inc. | Multiplex quantitative nucleic acid amplification and melting assay |
US9598739B1 (en) | 2009-03-13 | 2017-03-21 | Grifols Therapeutics Inc. | Human erythrovirus |
US8481698B2 (en) * | 2009-03-19 | 2013-07-09 | The President And Fellows Of Harvard College | Parallel proximity ligation event analysis |
EP2411543B1 (en) | 2009-03-27 | 2017-04-19 | Life Technologies Corporation | Methods, compositions, and kits for detecting allelic variants |
JP5985390B2 (en) | 2009-04-02 | 2016-09-06 | フリューダイム・コーポレイション | Multi-primer amplification method for adding barcode to target nucleic acid |
DE212010000039U1 (en) * | 2009-04-03 | 2012-02-02 | Helixis, Inc. | DEVICES FOR HEATING BIOLOGICAL SAMPLES |
US20100272824A1 (en) * | 2009-04-16 | 2010-10-28 | The Texas A&M University System | Compositions and methods for preventing and monitoring disease |
WO2010124257A2 (en) * | 2009-04-24 | 2010-10-28 | Colby Pharmaceutical Company | Methods and kits for determining oxygen free radical (ofr) levels in animal and human tissues as a prognostic marker for cancer and other pathophysiologies |
US9206482B2 (en) | 2009-04-29 | 2015-12-08 | Genomedx Biosciences Inc. | Systems and methods for expression-based classification of thyroid tissue |
EP2256215A1 (en) * | 2009-04-30 | 2010-12-01 | Steffen Mergemeier | Assay system using a nuclease activity of a nucleic acid polymerase |
CN102459636B (en) | 2009-05-07 | 2016-08-17 | 威拉赛特公司 | For diagnosing the method and composition of disorder of thyroid gland |
US20100299773A1 (en) * | 2009-05-20 | 2010-11-25 | Monsanto Technology Llc | Methods and compositions for selecting an improved plant |
CA2762986C (en) | 2009-05-22 | 2018-03-06 | Asuragen, Inc. | Mirna biomarkers of prostate disease |
WO2010138908A1 (en) | 2009-05-29 | 2010-12-02 | Ventana Medical Systems, Inc. | Igfir gene copy number as a prognostic marker in a non-small cell lung cancer |
JP2012528569A (en) | 2009-05-29 | 2012-11-15 | ヴェンタナ メディカル システムズ, インク. | A method for scoring gene copy number of biological samples using in situ hybridization |
AU2010265889A1 (en) | 2009-06-25 | 2012-01-19 | Yale University | Single nucleotide polymorphisms in BRCA1 and cancer risk |
CA2767616A1 (en) | 2009-07-09 | 2011-01-13 | The Scripps Research Institute | Gene expression profiles associated with chronic allograft nephropathy |
WO2011006161A2 (en) | 2009-07-10 | 2011-01-13 | The Regents Of The University Of Michigan | Compositions and methods for diagnosing and treating macular degeneration |
JP2012533545A (en) | 2009-07-14 | 2012-12-27 | ベス・イスラエル・ディーコネス・メディカル・センター,インコーポレイテッド | Methods for increasing liver growth |
EP2456884A1 (en) | 2009-07-22 | 2012-05-30 | E. I. Du Pont De Nemours And Company | Sequences and their use for detection and characterization of e. coli o157:h7 |
EP2462515A4 (en) | 2009-08-05 | 2015-08-12 | Life Technologies Corp | Methods for the analysis of proximity binding assay data |
US20110045458A1 (en) * | 2009-08-20 | 2011-02-24 | Mayo Foundation For Medical Education And Research | Detection of Enterovirus |
EP2470897A4 (en) | 2009-08-28 | 2013-05-29 | Asuragen Inc | Mirna biomarkers of lung disease |
EP2470184B1 (en) | 2009-08-28 | 2018-08-01 | Bellweather Farms | Chemical induction of lactation |
WO2011028539A1 (en) | 2009-09-02 | 2011-03-10 | Quantalife, Inc. | System for mixing fluids by coalescence of multiple emulsions |
GB0915664D0 (en) | 2009-09-08 | 2009-10-07 | Enigma Diagnostics Ltd | Reaction method |
EP2301666B1 (en) * | 2009-09-09 | 2021-06-30 | Cole-Parmer Ltd. | Optical system for multiple reactions |
US20110070590A1 (en) | 2009-09-22 | 2011-03-24 | Jan Rohozinski | Primers and Methods for Determining RhD Zygosity |
US8815515B1 (en) | 2009-09-24 | 2014-08-26 | University Of Utah Research Foundation | Methods, compositions, and kits for rare allele detection |
WO2011037802A2 (en) | 2009-09-28 | 2011-03-31 | Igor Kutyavin | Methods and compositions for detection of nucleic acids based on stabilized oligonucleotide probe complexes |
US8975019B2 (en) * | 2009-10-19 | 2015-03-10 | University Of Massachusetts | Deducing exon connectivity by RNA-templated DNA ligation/sequencing |
WO2011050938A1 (en) | 2009-10-26 | 2011-05-05 | Genovoxx Gmbh | Conjugates of nucleotides and method for the application thereof |
WO2011059686A2 (en) | 2009-10-29 | 2011-05-19 | Wisconsin Alumni Research Foundation | Detection of b-cell activating factor as a biomaker for antibody mediated rejection in transplant recipients |
WO2011053845A2 (en) | 2009-10-30 | 2011-05-05 | Illumina, Inc. | Microvessels, microparticles, and methods of manufacturing and using the same |
US20110117546A1 (en) * | 2009-11-18 | 2011-05-19 | Microfluidic Systems, Inc. | Increase of signal sensitivity using dual probes in pcr reactions |
EP3263124A1 (en) | 2009-11-20 | 2018-01-03 | Oregon Health&Science University | Methods for producing an immune response to tuberculosis |
JP2013511292A (en) | 2009-11-23 | 2013-04-04 | ベクトン・ディキンソン・アンド・カンパニー | Method for assaying a target nucleic acid by signal amplification using probe hybridization and restriction |
US9133343B2 (en) | 2009-11-30 | 2015-09-15 | Enzo Biochem, Inc. | Dyes and compositions, and processes for using same in analysis of protein aggregation and other applications |
WO2011066589A1 (en) | 2009-11-30 | 2011-06-03 | Caris Life Sciences Luxembourg Holdings | Methods and systems for isolating, storing, and analyzing vesicles |
CA2782692C (en) | 2009-12-03 | 2021-11-09 | Quest Diagnostics Investments Incorporated | Methods for the diagnosis of bacterial vaginosis |
EP2511370B1 (en) * | 2009-12-07 | 2016-09-07 | ARKRAY, Inc. | PROBE FOR DETECTING POLYMORPHISMS IN THE K-ras GENE, AND USE THEREOF |
US10446272B2 (en) | 2009-12-09 | 2019-10-15 | Veracyte, Inc. | Methods and compositions for classification of samples |
US8614071B2 (en) | 2009-12-11 | 2013-12-24 | Roche Molecular Systems, Inc. | Preferential amplification of mRNA over DNA using chemically modified primers |
CA2784344C (en) | 2009-12-21 | 2018-01-02 | Seegene, Inc. | Tsg primer target detection |
US8877437B1 (en) | 2009-12-23 | 2014-11-04 | Biotium, Inc. | Methods of using dyes in association with nucleic acid staining or detection |
WO2011082253A2 (en) | 2009-12-30 | 2011-07-07 | Board Of Trustees Of Michigan State University | A method to produce acetyldiacylglycerols (ac-tags) by expression ofan acetyltransferase gene isolated from euonymus alatus (burning bush) |
EP2524055A4 (en) | 2010-01-11 | 2013-06-05 | Genomic Health Inc | Method to use gene expression to determine likelihood of clinical outcome of renal cancer |
TWI535445B (en) | 2010-01-12 | 2016-06-01 | 安可美德藥物股份有限公司 | Wnt antagonists and methods of treatment and screening |
EP2524059A4 (en) | 2010-01-13 | 2013-11-20 | Caris Life Sciences Luxembourg Holdings | Detection of gastrointestinal disorders |
US8574832B2 (en) | 2010-02-03 | 2013-11-05 | Massachusetts Institute Of Technology | Methods for preparing sequencing libraries |
TWI518325B (en) | 2010-02-04 | 2016-01-21 | 自治醫科大學 | Identification, assessment, and therapy of cancers with innate or acquired resistance to alk inhibitors |
EP2531856A4 (en) * | 2010-02-05 | 2013-07-10 | Translational Genomics Res Inst | Methods and kits used in classifying adrenocortical carcinoma |
WO2011100057A2 (en) * | 2010-02-09 | 2011-08-18 | Eugene Spier | Methods and compositions for universal detection of nucleic acids |
CA2789456C (en) | 2010-02-16 | 2017-11-21 | Becton, Dickinson And Company | Assay for detecting closely-related serotypes of human papillomavirus (hpv) |
EP2542696B1 (en) | 2010-03-01 | 2016-09-28 | Caris Life Sciences Switzerland Holdings GmbH | Biomarkers for theranostics |
US8399198B2 (en) | 2010-03-02 | 2013-03-19 | Bio-Rad Laboratories, Inc. | Assays with droplets transformed into capsules |
US9458513B2 (en) | 2010-03-23 | 2016-10-04 | Wako Pure Chemical Industries, Ltd. | Primer and probe for detecting chlamydia trachomatis, and method for detecting chlamydia trachomatis using same |
EP2556170A4 (en) | 2010-03-25 | 2014-01-01 | Quantalife Inc | Droplet transport system for detection |
JP2013524169A (en) | 2010-03-25 | 2013-06-17 | クァンタライフ・インコーポレーテッド | Detection system for assay by droplet |
US9506057B2 (en) | 2010-03-26 | 2016-11-29 | Integrated Dna Technologies, Inc. | Modifications for antisense compounds |
EP2553123B1 (en) * | 2010-03-26 | 2016-08-24 | Integrated DNA Technologies, Inc. | Methods for enhancing nucleic acid hybridization |
CN102971337B (en) | 2010-04-01 | 2016-09-21 | 昂考梅德药品有限公司 | FZ combines medicament and application thereof |
US9125931B2 (en) | 2010-04-06 | 2015-09-08 | Massachusetts Institute Of Technology | Post-transcriptional regulation of RNA-related processes using encoded protein-binding RNA aptamers |
GB2491795A (en) | 2010-04-06 | 2012-12-12 | Massachusetts Inst Technology | Gene-expression profiling with reduced numbers of transcript measurements |
AU2011237669B2 (en) | 2010-04-06 | 2016-09-08 | Caris Life Sciences Switzerland Holdings Gmbh | Circulating biomarkers for disease |
DE102010003782B4 (en) | 2010-04-08 | 2023-09-28 | Ist Innuscreen Gmbh | Device for detecting nucleic acids |
DE102010003781B4 (en) | 2010-04-08 | 2012-08-16 | Aj Innuscreen Gmbh | Method for detecting specific nucleic acid sequences |
CN103038774B (en) | 2010-04-11 | 2017-09-22 | 生命技术公司 | The system and method for qPCR based on model |
US20110250598A1 (en) | 2010-04-12 | 2011-10-13 | Ulrike Fischer | Detergent free polymerases |
CN103079567A (en) | 2010-04-17 | 2013-05-01 | 拜尔健康护理有限责任公司 | Synthetic metabolites of fluoro substituted omega-carboxyaryl diphenyl urea for the treatment and prevention diseases and conditions |
US20110269735A1 (en) | 2010-04-19 | 2011-11-03 | Celera Corporation | Genetic polymorphisms associated with statin response and cardiovascular diseases, methods of detection and uses thereof |
EP3508854A1 (en) | 2010-04-27 | 2019-07-10 | The Regents of The University of California | Cancer biomarkers and methods of use thereof |
US9309565B2 (en) | 2010-05-14 | 2016-04-12 | Life Technologies Corporation | Karyotyping assay |
US8658776B2 (en) | 2010-05-28 | 2014-02-25 | Life Technologies Corporation | Synthesis of 2′,3′-dideoxynucleosides for automated DNA synthesis and pyrophosphorolysis activated polymerization |
EP3150723B1 (en) | 2010-06-21 | 2019-01-30 | Life Technologies Corporation | Compositions, kits, and methods for synthesis and/or detection of nucleic acids |
CN107760770B (en) | 2010-06-21 | 2022-05-17 | 生命技术公司 | Compositions, methods and kits for nucleic acid synthesis and amplification |
US9650629B2 (en) | 2010-07-07 | 2017-05-16 | Roche Molecular Systems, Inc. | Clonal pre-amplification in emulsion |
EP3674423A1 (en) | 2010-07-12 | 2020-07-01 | Gen-Probe Incorporated | Compositions and assays to detect seasonal h3 influenza a virus nucleic acid |
EP2407242A1 (en) | 2010-07-13 | 2012-01-18 | Dublin City University | Direct clone analysis and selection technology |
EP2752671A3 (en) | 2010-07-23 | 2016-08-24 | Beckman Coulter, Inc. | System or method of including analytical units |
US9046507B2 (en) | 2010-07-29 | 2015-06-02 | Gen-Probe Incorporated | Method, system and apparatus for incorporating capacitive proximity sensing in an automated fluid transfer procedure |
WO2012016936A1 (en) | 2010-07-31 | 2012-02-09 | Aj Innuscreen Gmbh | Detection of specific nucleic acid sequences by means of fluorescence quenching |
DE102010033107A1 (en) | 2010-08-02 | 2012-02-02 | Aj Innuscreen Gmbh | Detection of specific nucleic acid sequences by fluorescence quenching |
JP5887041B2 (en) | 2010-08-02 | 2016-03-16 | ウェイト、ブレント、エル. | Pressurizable cartridge for polymerase chain reaction |
JP5714016B2 (en) * | 2010-08-23 | 2015-05-07 | 株式会社島津製作所 | Switching type fluorescent nanoparticle probe and fluorescent molecular imaging method using the same |
US8557532B2 (en) | 2010-08-30 | 2013-10-15 | The University Of Utah Research Foundation | Diagnosis and treatment of drug-resistant Ewing'S sarcoma |
EP2611932A2 (en) | 2010-08-30 | 2013-07-10 | Gen-Probe Incorporated | Compositions, methods and reaction mixtures for the detection of xenotropic murine leukemia virus-related virus |
CA2809457C (en) | 2010-09-07 | 2019-07-30 | Integrated Dna Technologies, Inc. | Modifications for antisense compounds |
US9051606B2 (en) | 2010-09-10 | 2015-06-09 | Qiagen Gaithersburg, Inc. | Methods and compositions for nucleic acid detection |
WO2012038049A2 (en) | 2010-09-22 | 2012-03-29 | Roche Diagnostics Gmbh | Amplification of distant nucleic acid targets using engineered primers |
IN2013MN00522A (en) | 2010-09-24 | 2015-05-29 | Univ Leland Stanford Junior | |
WO2012042516A2 (en) | 2010-09-29 | 2012-04-05 | Mor Research Applications Ltd. | Prognostic methods, compositions and kits for prediction of acute lymphoblastic leukemia (all) relapse |
CA2810317A1 (en) | 2010-10-04 | 2012-04-12 | F. Hoffmann-La Roche Ag | Method for cell lysis in a pcr reaction buffer |
CA2810316A1 (en) | 2010-10-04 | 2012-04-12 | F. Hoffmann-La Roche Ag | Method for cell lysis and pcr within the same reaction vessel |
EP2625284B1 (en) | 2010-10-04 | 2015-01-28 | Roche Diagniostics GmbH | Method for cell lysis in a rt-pcr reaction buffer |
US20150218639A1 (en) | 2014-01-17 | 2015-08-06 | Northwestern University | Biomarkers predictive of predisposition to depression and response to treatment |
US20150225792A1 (en) | 2014-01-17 | 2015-08-13 | Northwestern University | Compositions and methods for identifying depressive disorders |
US10093981B2 (en) | 2010-10-19 | 2018-10-09 | Northwestern University | Compositions and methods for identifying depressive disorders |
US10233501B2 (en) | 2010-10-19 | 2019-03-19 | Northwestern University | Biomarkers predictive of predisposition to depression and response to treatment |
ES2576927T3 (en) | 2010-10-22 | 2016-07-12 | T2 Biosystems, Inc. | NMR systems and methods for rapid analyte detection |
KR20120042100A (en) | 2010-10-22 | 2012-05-03 | 주식회사 씨젠 | Detection of target nucleic acid sequences using dual-labeled immobilized probes on solid phase |
US8563298B2 (en) | 2010-10-22 | 2013-10-22 | T2 Biosystems, Inc. | NMR systems and methods for the rapid detection of analytes |
CN103429331B (en) | 2010-11-01 | 2016-09-28 | 伯乐生命医学产品有限公司 | For forming the system of emulsion |
US20120108651A1 (en) | 2010-11-02 | 2012-05-03 | Leiden University Medical Center (LUMC) Acting on Behalf of Academic Hospital Leiden (AZL) | Genetic polymorphisms associated with venous thrombosis and statin response, methods of detection and uses thereof |
EP2640366A2 (en) | 2010-11-15 | 2013-09-25 | Exelixis, Inc. | Benzoxazepines as inhibitors of pi3k/mtor and methods of their use and manufacture |
EP2640367A2 (en) | 2010-11-15 | 2013-09-25 | Exelixis, Inc. | Benzoxazepines as inhibitors of pi3k/mtor and methods of their use and manufacture |
US20130304390A1 (en) | 2010-11-16 | 2013-11-14 | Life Technologies Corporation | Systems and Methods for the Analysis of Proximity Binding Assay Data |
DE102010052524A1 (en) | 2010-11-22 | 2012-05-24 | Aj Innuscreen Gmbh | Method for the qualitative and quantitative detection of specific nucleic acid sequences in real time |
AU2010364322C1 (en) | 2010-11-24 | 2013-09-19 | E. I. Du Pont De Nemours And Company | Brassica GAT event DP-073496-4 and compositions and methods for the identification and/or detection thereof |
WO2012071509A2 (en) | 2010-11-24 | 2012-05-31 | Exelixis, Inc. | Benzoxazepines as inhibitors of p13k/mtor and methods of their use and manufacture |
US9145592B2 (en) | 2010-11-24 | 2015-09-29 | Alina Cywinska | Qualitative/quantitative detection of fungal species |
WO2012071039A1 (en) | 2010-11-24 | 2012-05-31 | Pioner Hi-Bred International, Inc. | Brassica gat event dp-061061-7 and compositions and methods for the identification and/or detection thereof |
WO2012073053A1 (en) | 2010-11-30 | 2012-06-07 | Diagon Kft. | Procedure for nucleic acid-based molecular diagnostic determination of bacterial germ counts and kit for this purpose |
EP2649202B1 (en) * | 2010-12-07 | 2019-02-20 | Bio-Rad Laboratories, Inc. | Nucleic acid target detection using a detector, a probe and an inhibitor |
CA2858526C (en) | 2010-12-07 | 2020-06-30 | Northwestern University | Molecular targets for als and related disorders |
TWI667347B (en) | 2010-12-15 | 2019-08-01 | 瑞士商先正達合夥公司 | Soybean event syht0h2 and compositions and methods for detection thereof |
EP3839064A1 (en) | 2010-12-27 | 2021-06-23 | Abbott Molecular Inc. | Systems for quantitating high titer samples by digital pcr |
CN107326073A (en) | 2010-12-29 | 2017-11-07 | 生命技术公司 | It is used as the DDAO compounds of fluorescence reference standard items |
BR112013015595A2 (en) | 2010-12-30 | 2017-06-20 | Pioneer Hi Bred Int | method of identifying a first soybean or germplasm plant that exhibits enhanced resistance to one or more soybean aphid biotypes, isolated polynucleotide, kit for detecting or selecting at least one improved aphid-resistant soybean plant |
MX2017015093A (en) | 2011-01-11 | 2023-03-10 | Seegene Inc | Detection of target nucleic acid sequences by pto cleavage and extension assay. |
EP3733870A3 (en) | 2011-01-14 | 2021-01-27 | Life Technologies Corporation | Methods for identification and quantification of mirnas |
DK2665833T3 (en) | 2011-01-17 | 2017-07-24 | Life Technologies Corp | WORKING PROCEDURE FOR DETECTING LIGANDS USING NUCLEIC ACIDS |
KR20140006898A (en) | 2011-01-25 | 2014-01-16 | 알막 다이아그노스틱스 리미티드 | Colon cancer gene expression signatures and methods of use |
US9127309B2 (en) | 2011-01-31 | 2015-09-08 | Qiagen Mansfield, Inc. | Methods of nucleic acid quantification and detection using anomalous migration |
US20140024590A1 (en) | 2011-02-18 | 2014-01-23 | Yale University | KRAS-Variant And Endometriosis |
CN103403533B (en) | 2011-02-24 | 2017-02-15 | 简.探针公司 | Systems and methods for distinguishing optical signals of different modulation frequencies in an optical signal detector |
PE20140627A1 (en) | 2011-03-02 | 2014-05-30 | Berg Llc | CELL-BASED INTERROGATORY TESTS AND THE USE OF THEM |
EP2686449B1 (en) | 2011-03-18 | 2020-11-18 | Bio-Rad Laboratories, Inc. | Multiplexed digital assays with combinatorial use of signals |
WO2012129347A1 (en) | 2011-03-21 | 2012-09-27 | Biodesy, Llc | Classification of kinase inhibitors using nonlinear optical techniques |
EP2689030A1 (en) | 2011-03-21 | 2014-01-29 | Yale University | The kras variant and tumor biology |
US11078525B2 (en) | 2011-03-29 | 2021-08-03 | Seegene, Inc. | Detection of target nucleic acid sequence by PTO cleavage and extension-dependent cleavage |
US20120328567A1 (en) | 2011-04-08 | 2012-12-27 | Steven Bushnell | Biomarkers predictive of therapeutic responsiveness to ifnb and uses thereof |
ES2769028T3 (en) | 2011-04-15 | 2020-06-24 | Becton Dickinson Co | Real-time scanning microfluidic thermocycler |
WO2012142003A2 (en) | 2011-04-15 | 2012-10-18 | Life Technologies Corporation | Chemical ligation |
EP2702175B1 (en) | 2011-04-25 | 2018-08-08 | Bio-Rad Laboratories, Inc. | Methods and compositions for nucleic acid analysis |
WO2012146377A1 (en) | 2011-04-27 | 2012-11-01 | Dmitry Cherkasov | Method and components for detecting nucleic acid chains |
US10172305B2 (en) | 2011-04-29 | 2019-01-08 | Monsanto Technology Llc | Diagnostic molecular markers for seed lot purity traits in soybeans |
US9708672B2 (en) | 2011-05-02 | 2017-07-18 | Nutech Ventures | Plants with useful traits and related methods |
MX342067B (en) | 2011-05-04 | 2016-09-09 | Seegene Inc | Detection of target nucleic acid sequences by po cleavage and hybridization. |
US9850524B2 (en) | 2011-05-04 | 2017-12-26 | Seegene, Inc. | Detection of target nucleic acid sequences by PO cleavage and hybridization |
EP2704688B1 (en) | 2011-05-05 | 2019-07-10 | Matinas BioPharma Nanotechnologies, Inc. | Cochleate compositions and methods of making and using same |
EP2707496A1 (en) | 2011-05-11 | 2014-03-19 | Diagon Kft. | Procedure for rapid determination of viruses using nucleic acid-based molecular diagnostics, and a kit for this purpose |
WO2012170907A2 (en) | 2011-06-08 | 2012-12-13 | Life Technologies Corporation | Polymerization of nucleic acids using proteins having low isoelectric points |
CN106518696B (en) | 2011-06-08 | 2019-06-14 | 生命技术公司 | The design and exploitation of novel detergent for PCR system |
WO2012171997A1 (en) | 2011-06-14 | 2012-12-20 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods for determining the expression level of a gene of interest including correction of rt-qpcr data for genomic dna-derived signals |
US8815509B2 (en) * | 2011-06-14 | 2014-08-26 | University Of Southern California | Fluorescence-based assay for the rapid detection and quantification of deoxyribonucleoside triphosphates |
WO2013003350A2 (en) | 2011-06-27 | 2013-01-03 | Eisai R&D Management Co., Ltd. | Microrna biomarkers indicative of alzheimer's disease |
US9487824B2 (en) | 2011-06-28 | 2016-11-08 | Igor Kutyavin | Methods and compositions for enrichment of nucleic acids in mixtures of highly homologous sequences |
CA2838091C (en) | 2011-07-05 | 2019-08-13 | The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services, Centers For Disease Control And Prevention | Hiv-1 genotyping assay for global surveillance of hiv-1 drug resistance |
EP2737089B1 (en) | 2011-07-29 | 2017-09-06 | Bio-rad Laboratories, Inc. | Library characterization by digital assay |
US9758837B2 (en) | 2011-08-02 | 2017-09-12 | The United States Of America, As Represented By The Secretary Of Agriculture | Sensitive and rapid method for Candidatus Liberibacter species detection |
US8759618B2 (en) | 2011-08-17 | 2014-06-24 | Stine Seed Farm, Inc. | Maize event HCEM485, compositions and methods for detecting and use thereof |
JP2014526892A (en) | 2011-08-18 | 2014-10-09 | ネステク ソシエテ アノニム | Compositions and methods for detecting allelic polymorphisms |
BR112014004213A2 (en) | 2011-08-23 | 2017-06-20 | Found Medicine Inc | new kif5b-ret fusion molecules and their uses |
US20140228243A1 (en) | 2011-09-04 | 2014-08-14 | Yissum Research Development Company Of The Hebrew University Of Jerusalem | Prognostic methods and compositions for predicting interferon treatment eficacy in a subject |
US9644241B2 (en) | 2011-09-13 | 2017-05-09 | Interpace Diagnostics, Llc | Methods and compositions involving miR-135B for distinguishing pancreatic cancer from benign pancreatic disease |
WO2013040491A2 (en) | 2011-09-15 | 2013-03-21 | Shafer David A | Probe: antiprobe compositions for high specificity dna or rna detection |
LT2709613T (en) | 2011-09-16 | 2018-02-12 | Gilead Pharmasset Llc | Methods for treating hcv |
WO2013041577A1 (en) | 2011-09-20 | 2013-03-28 | Vib Vzw | Methods for the diagnosis of amyotrophic lateral sclerosis and frontotemporal lobar degeneration |
LT2768607T (en) | 2011-09-26 | 2021-12-27 | Thermo Fisher Scientific Geneart Gmbh | Multiwell plate for high efficiency, small volume nucleic acid synthesis |
WO2014153188A2 (en) | 2013-03-14 | 2014-09-25 | Life Technologies Corporation | High efficiency, small volume nucleic acid synthesis |
EP2761023B1 (en) | 2011-09-28 | 2018-03-07 | Qualicon Diagnostics LLC | Sequences and their use for detection and characterization of stec bacteria |
CN108192952A (en) | 2011-09-29 | 2018-06-22 | 露美内克丝公司 | Hydrolysis probes |
US9416153B2 (en) | 2011-10-11 | 2016-08-16 | Enzo Life Sciences, Inc. | Fluorescent dyes |
EP2773892B1 (en) | 2011-11-04 | 2020-10-07 | Handylab, Inc. | Polynucleotide sample preparation device |
WO2013070748A1 (en) | 2011-11-07 | 2013-05-16 | Beckman Coulter, Inc. | Magnetic damping for specimen transport system |
WO2013070756A2 (en) | 2011-11-07 | 2013-05-16 | Beckman Coulter, Inc. | System and method for processing samples |
BR112014011048A2 (en) | 2011-11-07 | 2017-05-02 | Beckman Coulter Inc | robotic arm |
CN104105969B (en) | 2011-11-07 | 2016-10-12 | 贝克曼考尔特公司 | Centrifuge system and workflow |
BR112014011035A2 (en) | 2011-11-07 | 2017-06-13 | Beckman Coulter, Inc. | aliquot system and workflow |
US9046506B2 (en) | 2011-11-07 | 2015-06-02 | Beckman Coulter, Inc. | Specimen container detection |
US20130115609A1 (en) * | 2011-11-08 | 2013-05-09 | National Cheng Kung University | Methods and Kits for Detecting Circulating Cancer Stem Cells |
BR112014011094A2 (en) | 2011-11-09 | 2018-08-07 | Du Pont | method for detecting salmonella, isolated polynucleotide, kit and reagent tablet |
US20130179086A1 (en) | 2011-11-29 | 2013-07-11 | Life Technologies Corporation | Systems and methods for the determination of a copy number of a genomic sequence |
US8889159B2 (en) | 2011-11-29 | 2014-11-18 | Gilead Pharmasset Llc | Compositions and methods for treating hepatitis C virus |
US8663924B2 (en) | 2011-11-30 | 2014-03-04 | Agilent Technologies, Inc. | Quantitative PCR-based method to predict the efficiency of target enrichment for next-generation sequencing using repetitive DNA elements (lines/sines) as negative controls |
EP2791359B1 (en) | 2011-12-13 | 2020-01-15 | Decipher Biosciences, Inc. | Cancer diagnostics using non-coding transcripts |
WO2013096798A2 (en) | 2011-12-22 | 2013-06-27 | Ibis Biosciences, Inc. | Amplification of a sequence from a ribonucleic acid |
CA2862269C (en) | 2011-12-29 | 2021-09-14 | Pioneer Hi-Bred International, Inc. | Methods of improving aphid resistance in soybeans |
WO2013101741A1 (en) | 2011-12-30 | 2013-07-04 | Abbott Molecular, Inc. | Channels with cross-sectional thermal gradients |
WO2013112458A1 (en) | 2012-01-24 | 2013-08-01 | Beth Israel Deaconess Medical Center, Inc. | Novel chrebp isoforms and methods using the same |
KR20130101952A (en) | 2012-02-02 | 2013-09-16 | 주식회사 씨젠 | Detection of target nucleic acid sequence by pto cleavage and extension-dependent hybridization |
KR102145677B1 (en) | 2012-02-03 | 2020-08-18 | 캘리포니아 인스티튜트 오브 테크놀로지 | Signal encoding and decoding in multiplexed biochemical assays |
WO2013126793A2 (en) | 2012-02-24 | 2013-08-29 | Gen-Probe Prodesse, Inc. | Detection of shiga toxin genes in bacteria |
US9045803B2 (en) | 2012-02-29 | 2015-06-02 | Abbott Molecular Inc. | Hepatitis B virus typing and resistance assay |
ES2669244T3 (en) | 2012-03-05 | 2018-05-24 | Seegene, Inc. | Detection of nucleotide variation in a target nucleic acid sequence by CTO cleavage and extension assay |
WO2013139860A1 (en) | 2012-03-21 | 2013-09-26 | Roche Diagnostics Gmbh | Methods for assessing rna quality |
US9133509B2 (en) | 2012-03-22 | 2015-09-15 | Lgc Genomics Limited | Polymerase chain reaction detection system |
US9682093B2 (en) | 2012-03-30 | 2017-06-20 | Charles R. Drew University Of Medicine And Science | Compositions and methods for treating or preventing metabolic syndrome disorders |
BR112014024537A2 (en) | 2012-04-02 | 2017-08-08 | Berg Llc | methods to identify modulators of a biological system, a disease process, and angiogenesis |
WO2013155223A1 (en) | 2012-04-10 | 2013-10-17 | The Regents Of The University Of California | Compositions and methods for treating cancer |
WO2013168162A1 (en) | 2012-05-09 | 2013-11-14 | Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. | Clustered single nucleotide polymorphisms in the human acetylcholinesterase gene and uses thereof in diagnosis and therapy |
MX361457B (en) | 2012-05-11 | 2018-12-06 | Reset Therapeutics Inc | Carbazole-containing sulfonamides as cryptochrome modulators. |
CN104471077B (en) | 2012-05-21 | 2017-05-24 | 富鲁达公司 | Single-particle analysis of particle populations |
ES2770638T3 (en) | 2012-06-14 | 2020-07-02 | Life Technologies Corp | New compositions, methods and kits for polymerase chain reaction (PCR) |
US9347105B2 (en) | 2012-06-15 | 2016-05-24 | Pioneer Hi Bred International Inc | Genetic loci associated with resistance of soybean to cyst nematode and methods of use |
WO2013192616A1 (en) | 2012-06-22 | 2013-12-27 | Htg Molecular Diagnostics, Inc. | Molecular malignancy in melanocytic lesions |
US9657290B2 (en) | 2012-07-03 | 2017-05-23 | The Board Of Trustees Of The Leland Stanford Junior University | Scalable bio-element analysis |
IN2015DN01609A (en) | 2012-08-03 | 2015-07-03 | California Inst Of Techn | |
US11035005B2 (en) | 2012-08-16 | 2021-06-15 | Decipher Biosciences, Inc. | Cancer diagnostics using biomarkers |
CN104769134A (en) | 2012-09-11 | 2015-07-08 | 赛拉诺斯股份有限公司 | Information management systems and methods using a biological signature |
WO2014051076A1 (en) | 2012-09-28 | 2014-04-03 | 株式会社Bna | Bna clamp method |
WO2014055746A1 (en) | 2012-10-04 | 2014-04-10 | The Board Of Trustees Of The Leland Stanford Junior University | Methods and reagents for detection, quantitation, and serotyping of dengue viruses |
US20160046997A1 (en) | 2012-10-18 | 2016-02-18 | Oslo Universitetssykehus Hf | Biomarkers for cervical cancer |
WO2014066328A1 (en) | 2012-10-23 | 2014-05-01 | Oncomed Pharmaceuticals, Inc. | Methods of treating neuroendocrine tumors using wnt pathway-binding agents |
DK2914741T3 (en) | 2012-11-02 | 2017-11-20 | Life Technologies Corp | New Compositions and Methods for Improving PCR Specificity |
EP2914621B1 (en) | 2012-11-05 | 2023-06-07 | Foundation Medicine, Inc. | Novel ntrk1 fusion molecules and uses thereof |
US10314253B2 (en) | 2012-12-04 | 2019-06-11 | Seminis Vegetable Seeds, Inc. | Methods and compositions for watermelon sex expression |
EP2929051B1 (en) | 2012-12-05 | 2018-01-10 | Amplidiag Oy | Method for detecting helicobacter pylori dna in a stool sample |
AU2013205090B2 (en) | 2012-12-07 | 2016-07-28 | Gen-Probe Incorporated | Compositions and Methods for Detecting Gastrointestinal Pathogen Nucleic Acid |
KR101760267B1 (en) | 2012-12-27 | 2017-07-24 | 주식회사 씨젠 | Detection of Target Nucleic Acid Sequence by PTO Cleavage and Extension-Dependent Non-Hybridization Assay |
US9856474B2 (en) | 2013-01-16 | 2018-01-02 | Iowa State University Research Foundation, Inc. | Deep intronic target for splicing correction on spinal muscular atrophy gene |
EP3939614A1 (en) | 2013-01-18 | 2022-01-19 | Foundation Medicine, Inc. | Methods of treating cholangiocarcinoma |
PE20151778A1 (en) | 2013-01-31 | 2015-12-16 | Gilead Pharmasset Llc | COMBINED FORMULATION OF TWO ANTIVIRAL COMPOUNDS |
CN105073195A (en) | 2013-02-04 | 2015-11-18 | 昂科梅德制药有限公司 | Methods and monitoring of treatment with a Wnt pathway inhibitor |
EP2959012B1 (en) | 2013-02-21 | 2018-02-14 | Qualicon Diagnostics LLC | Sequences and their use for detection and characterization of e. coli o157:h7 |
ES2662825T3 (en) | 2013-02-25 | 2018-04-09 | Seegene, Inc. | Detection of nucleotide variation in a sequence of target nucleic acids |
US20140249037A1 (en) | 2013-03-04 | 2014-09-04 | Fry Laboratories, LLC | Method and kit for characterizing microorganisms |
EP3640347A3 (en) | 2013-03-12 | 2020-07-29 | Life Technologies Corporation | Universal reporter-based genotyping methods and materials |
WO2014142575A1 (en) | 2013-03-13 | 2014-09-18 | Seegene, Inc. | Quantification of target nucleic acid using melting peak analysis |
AU2013202805B2 (en) | 2013-03-14 | 2015-07-16 | Gen-Probe Incorporated | System and method for extending the capabilities of a diagnostic analyzer |
US9168300B2 (en) | 2013-03-14 | 2015-10-27 | Oncomed Pharmaceuticals, Inc. | MET-binding agents and uses thereof |
AU2014227883B9 (en) | 2013-03-15 | 2020-09-10 | Life Technologies Corporation | Classification and actionability indices for lung cancer |
US9227978B2 (en) | 2013-03-15 | 2016-01-05 | Araxes Pharma Llc | Covalent inhibitors of Kras G12C |
US10294489B2 (en) | 2013-03-15 | 2019-05-21 | Board Of Trustees Of Southern Illinois University | Soybean resistant to cyst nematodes |
EP2978862A4 (en) | 2013-03-26 | 2016-11-02 | Genetag Technology Inc | Dual probe:antiprobe compositions for dna and rna detection |
EP2981621B1 (en) | 2013-04-05 | 2019-05-22 | Life Technologies Corporation | Gene fusions |
EP2992112B1 (en) | 2013-04-22 | 2020-06-03 | Icahn School of Medicine at Mount Sinai | Mutations in pdgfrb and notch3 as causes of autosomal dominant infantile myofibromatosis |
US9896717B2 (en) | 2013-05-09 | 2018-02-20 | Bio-Rad Laboratories, Inc. | Magnetic immuno digital PCR assay |
WO2014189843A1 (en) | 2013-05-20 | 2014-11-27 | Board Of Trustees Of The University Of Arkansas | Gep5 model for multiple myeloma |
EP3000088A4 (en) | 2013-05-23 | 2018-06-27 | Iphenotype LLC | Method and system for maintaining or improving wellness |
US10059999B2 (en) | 2013-06-10 | 2018-08-28 | Monsanto Technology Llc | Molecular markers associated with soybean tolerance to low iron growth conditions |
EP3521452B1 (en) | 2013-06-19 | 2021-08-04 | Luminex Corporation | Real-time multiplexed hydrolysis probe assay |
WO2015008985A1 (en) | 2013-07-15 | 2015-01-22 | Seegene, Inc. | Detection of target nucleic acid sequence by pto cleavage and extension-dependent immobilized oligonucleotide hybridization |
CN105473737B (en) | 2013-07-25 | 2019-10-25 | 德诚分子诊断 | Method and composition for detection bacterium pollution |
EP3030680B1 (en) | 2013-08-09 | 2018-10-03 | Luminex Corporation | Melt discrimination and multiplexing in nucleic acid assays |
CA2920636A1 (en) | 2013-08-20 | 2015-02-23 | E. I. Du Pont De Nemours And Company | Sequences and their use for detection of salmonella enteritidis and/or salmonella typhimurium |
US20150132743A1 (en) | 2013-09-04 | 2015-05-14 | Fluidigm Corporation | Proximity assays for detecting nucleic acids and proteins in a single cell |
WO2015179773A1 (en) | 2014-05-22 | 2015-11-26 | The Scripps Research Institute | Tissue molecular signatures of kidney transplant rejections |
CN105722996A (en) | 2013-09-13 | 2016-06-29 | 生命科技公司 | Classification and actionability indices for cancer |
US10767188B2 (en) | 2013-09-25 | 2020-09-08 | Nutech Ventures | Methods and compositions for obtaining useful plant traits |
US10584387B2 (en) | 2013-10-09 | 2020-03-10 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Detection of hepatitis delta virus (HDV) for the diagnosis and treatment of Sjögren's syndrome and lymphoma |
RU2016117929A (en) | 2013-10-09 | 2017-11-15 | Флюоресентрик, Инк. | MULTIPLEX PROBES |
JO3805B1 (en) | 2013-10-10 | 2021-01-31 | Araxes Pharma Llc | Inhibitors of kras g12c |
WO2015057008A2 (en) | 2013-10-18 | 2015-04-23 | Seegene, Inc. | Detection of target nucleic acid sequence on solid phase by pto cleavage and extension using hcto assay |
CA2923296A1 (en) | 2013-10-25 | 2015-04-30 | Pioneer Hi-Bred International, Inc. | Stem canker tolerant soybeans and methods of use |
US9914964B2 (en) | 2013-10-25 | 2018-03-13 | Life Technologies Corporation | Compounds for use in PCR systems and applications thereof |
EP3063317B1 (en) * | 2013-10-28 | 2020-06-03 | DOTS Technology Corp. | Allergen detection |
EP3063297A1 (en) | 2013-11-01 | 2016-09-07 | Yissum Research Development Company of The Hebrew University of Jerusalem Ltd. | Diagnostic methods and kits for determining a personalized treatment regimen for a subject suffering from a pathologic disorder |
US10072288B2 (en) | 2013-11-11 | 2018-09-11 | Roche Molecular Systems, Inc. | Detecting single nucleotide polymorphism using overlapped primer and melting probe |
WO2015073709A2 (en) | 2013-11-14 | 2015-05-21 | The United States Of America, As Represented By The Secretary, Department Of Health & Human Services | Detection of atherosclerotic cardiovascular disease risk and heart failure risk |
NZ784204A (en) | 2013-11-27 | 2022-10-28 | Seminis Vegetable Seeds Inc | Disease resistance loci in onion |
EP3797595B1 (en) | 2013-12-04 | 2023-08-16 | Newleaf Symbiotics, Inc. | Compositions for treating plants |
US9909181B2 (en) | 2013-12-13 | 2018-03-06 | Northwestern University | Biomarkers for post-traumatic stress states |
US20150191794A1 (en) | 2014-01-05 | 2015-07-09 | Biomirna Holdings Ltd. | Lung Cancer Determinations Using MIRNA |
US10398772B2 (en) | 2014-01-08 | 2019-09-03 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Ras pathways as markers of protection against HIV and methods to improve vaccine efficacy |
US10392629B2 (en) | 2014-01-17 | 2019-08-27 | Board Of Trustees Of Michigan State University | Increased caloric and nutritional content of plant biomass |
US10619219B2 (en) * | 2014-02-07 | 2020-04-14 | University Of Iowa Research Foundation | Oligonucleotide-based probes and methods for detection of microbes |
US10619210B2 (en) | 2014-02-07 | 2020-04-14 | The Johns Hopkins University | Predicting response to epigenetic drug therapy |
EP2986761B1 (en) | 2014-02-13 | 2018-08-15 | Bio-rad Laboratories, Inc. | Chromosome conformation capture in droplet partitions |
BR112016019007B1 (en) | 2014-02-21 | 2022-05-17 | Syngenta Participations Ag | Methods for producing a maize plant expressing vip3a or maize germplasm with increased male fertility, breeding program, method for improving seed production of a maize plant expressing vip3a, and seed production program |
GB201403076D0 (en) * | 2014-02-21 | 2014-04-09 | ALERE TECHNOLOGIES GmbH | Methods for detecting multiple nucleic acids in a sample |
EP3757224A3 (en) | 2014-02-27 | 2021-03-17 | Biogen MA Inc. | Method of assessing risk of pml |
NZ630710A (en) | 2014-02-27 | 2016-03-31 | Seminis Vegetable Seeds Inc | Compositions and methods for peronospora resistance in spinach |
CA2943056A1 (en) | 2014-03-17 | 2015-09-24 | Newleaf Symbiotics, Inc. | Compositions and methods for improving tomato production |
WO2015149034A2 (en) | 2014-03-27 | 2015-10-01 | Life Technologies Corporation | Gene fusions and gene variants associated with cancer |
WO2015147370A1 (en) | 2014-03-28 | 2015-10-01 | Seegene, Inc. | Detection of target nucleic acid sequences using different detection temperatures |
US10844436B2 (en) | 2014-04-01 | 2020-11-24 | Cornell University | Use of double-stranded DNA in exosomes: a novel biomarker in cancer detection |
TWI690521B (en) | 2014-04-07 | 2020-04-11 | 美商同步製藥公司 | Carbazole-containing amides, carbamates, and ureas as cryptochrome modulators |
CL2015000980A1 (en) | 2014-04-18 | 2016-07-01 | Upstartdna Co Ltd Standard Entity | Methods to detect pathogens in cold water fish. |
WO2015172083A1 (en) | 2014-05-08 | 2015-11-12 | Biogen Ma Inc. | Dimethylfumarate and prodrugs for treatment of multiple sclerosis |
EP2942400A1 (en) | 2014-05-09 | 2015-11-11 | Lifecodexx AG | Multiplex detection of DNA that originates from a specific cell-type |
US10801067B2 (en) | 2014-05-09 | 2020-10-13 | Eurofins Lifecodexx Gmbh | Detection of DNA that originates from a specific cell-type and related methods |
EP2942401A1 (en) | 2014-05-09 | 2015-11-11 | Lifecodexx AG | Detection of DNA that originates from a specific cell-type |
US10443100B2 (en) | 2014-05-22 | 2019-10-15 | The Scripps Research Institute | Gene expression profiles associated with sub-clinical kidney transplant rejection |
US11104951B2 (en) | 2014-05-22 | 2021-08-31 | The Scripps Research Institute | Molecular signatures for distinguishing liver transplant rejections or injuries |
WO2015179777A2 (en) | 2014-05-22 | 2015-11-26 | The Scripps Research Institute | Gene expression profiles associated with sub-clinical kidney transplant rejection |
EP3825418A3 (en) | 2014-05-22 | 2021-09-15 | The Scripps Research Institute | Molecular signatures for distinguishing liver transplant rejections or injuries |
US10316369B2 (en) | 2014-06-27 | 2019-06-11 | Seminis Vegetable Seeds, Inc. | Methods and assays for male sterile watermelon |
BR112016030008A2 (en) | 2014-06-27 | 2017-10-24 | Abbott Lab | method for detecting human pegivirus 2 infection in an individual, for detecting human pegivirus 2 nucleic acid and for detecting human pegivirus 2 in a sample, and, composition |
EP4015649A1 (en) | 2014-07-10 | 2022-06-22 | Fluoresentric, Inc. | Dna amplification technology |
EP3175001B1 (en) | 2014-07-30 | 2021-02-10 | Mor Research Applications Ltd. | Prognostic methods and systems of treatment for acute lymphoblastic leukemia |
US9982291B2 (en) | 2014-08-11 | 2018-05-29 | Luminex Corporation | Probes for improved melt discrimination and multiplexing in nucleic acid assays |
JP6940404B2 (en) | 2014-08-19 | 2021-09-29 | エフ.ホフマン−ラ ロシュ アーゲーF. Hoffmann−La Roche Aktiengesellschaft | Methods and compositions for nucleic acid detection |
WO2016027168A2 (en) | 2014-08-20 | 2016-02-25 | Lifesplice Pharma Llc | Splice modulating oligonucleotides and methods of use thereof |
JO3556B1 (en) | 2014-09-18 | 2020-07-05 | Araxes Pharma Llc | Combination therapies for treatment of cancer |
JP2017528498A (en) | 2014-09-25 | 2017-09-28 | アラクセス ファーマ エルエルシー | Inhibitors of KRAS G12C mutant protein |
WO2016049568A1 (en) | 2014-09-25 | 2016-03-31 | Araxes Pharma Llc | Methods and compositions for inhibition of ras |
US10370673B2 (en) | 2014-09-26 | 2019-08-06 | Purecircle Sdn Bhd | Single nucleotide polymorphism (SNP) markers for stevia |
GB201417499D0 (en) | 2014-10-03 | 2014-11-19 | Convergence Pharmaceuticals | Novel use |
GB201417500D0 (en) | 2014-10-03 | 2014-11-19 | Convergence Pharmaceuticals | Novel use |
GB201417497D0 (en) | 2014-10-03 | 2014-11-19 | Convergence Pharmaceuticals | Novel use |
EP3005862A1 (en) | 2014-10-10 | 2016-04-13 | Seminis Vegetable Seeds, Inc. | Melon plants with improved disease tolerance |
WO2016061111A1 (en) | 2014-10-13 | 2016-04-21 | Life Technologies Corporation | Methods, kits & compositions for determining gene copy numbers |
AU2015336079C1 (en) | 2014-10-20 | 2020-12-17 | Gen-Probe Incorporated | Red blood cell lysis solution |
US20170335396A1 (en) | 2014-11-05 | 2017-11-23 | Veracyte, Inc. | Systems and methods of diagnosing idiopathic pulmonary fibrosis on transbronchial biopsies using machine learning and high dimensional transcriptional data |
WO2016090323A1 (en) | 2014-12-05 | 2016-06-09 | Prelude, Inc. | Dcis recurrence and invasive breast cancer |
SG11201704660YA (en) | 2014-12-08 | 2017-07-28 | Berg Llc | Use of markers including filamin a in the diagnosis and treatment of prostate cancer |
WO2016094330A2 (en) | 2014-12-08 | 2016-06-16 | 20/20 Genesystems, Inc | Methods and machine learning systems for predicting the liklihood or risk of having cancer |
JP6734851B2 (en) | 2014-12-09 | 2020-08-05 | シージーン アイエヌシー | Discrimination of signals for target nucleic acid sequences |
CN107532129B (en) | 2014-12-09 | 2022-09-13 | 生命技术公司 | Efficient small volume nucleic acid synthesis |
WO2016092045A1 (en) | 2014-12-11 | 2016-06-16 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods and kits for predicting medically refractory acute severe colitis |
EP3230467A1 (en) | 2014-12-12 | 2017-10-18 | ELITechGroup B.V. | Methods and compositions for detecting antibiotic resistant bacteria |
EP3230468B1 (en) | 2014-12-12 | 2020-09-16 | ELITechGroup, Inc. | Methods and kits for detecting antibiotic resistant bacteria |
WO2016106286A1 (en) | 2014-12-23 | 2016-06-30 | Biodesy, Inc. | Attachment of proteins to interfaces for use in nonlinear optical detection |
EP3037545A1 (en) | 2014-12-23 | 2016-06-29 | The Provost, Fellows, Foundation Scholars, & the other members of Board, of the College of the Holy & Undiv. Trinity of Queen Elizabeth near Dublin | A DNA-methylation test for prostate cancer |
US10370653B2 (en) | 2015-02-22 | 2019-08-06 | The Board Of Trustees Of The Leland Stanford Junior University | Micro-screening apparatus, process, and products |
EP3929291A1 (en) | 2015-03-17 | 2021-12-29 | Bio-Rad Laboratories, Inc. | Detection of genome editing |
US9708647B2 (en) | 2015-03-23 | 2017-07-18 | Insilixa, Inc. | Multiplexed analysis of nucleic acid hybridization thermodynamics using integrated arrays |
US9957393B2 (en) | 2015-03-30 | 2018-05-01 | Enzo Biochem, Inc. | Monoazo dyes with cyclic amine as fluorescence quenchers |
CN107849022A (en) | 2015-04-10 | 2018-03-27 | 亚瑞克西斯制药公司 | Substituted quinazoline compound and its application method |
CN104845967B (en) | 2015-04-15 | 2020-12-11 | 苏州新海生物科技股份有限公司 | Oligonucleotide fragment, method for selectively amplifying target nucleic acid sequence variant by using same and application of oligonucleotide fragment |
WO2016168540A1 (en) | 2015-04-15 | 2016-10-20 | Araxes Pharma Llc | Fused-tricyclic inhibitors of kras and methods of use thereof |
AU2016252998B2 (en) | 2015-04-24 | 2021-11-04 | Atila Biosystems Incorporated | Amplification with primers of limited nucleotide composition |
CN114574612A (en) | 2015-04-28 | 2022-06-03 | 孟山都技术有限公司 | Methods and compositions for producing short shoot corn plants |
CA2981819A1 (en) | 2015-04-30 | 2016-11-03 | Monsanto Technology Llc | Methods for producing canola plants with clubroot resistance and compositions thereof |
WO2016177774A1 (en) | 2015-05-04 | 2016-11-10 | Academisch Medisch Centrum | Method of quantifying mirnas using normalization |
CN108271342B (en) | 2015-06-10 | 2021-12-07 | 新叶共生有限公司 | Antifungal methylobacterium compositions and methods of use |
WO2016205233A2 (en) | 2015-06-15 | 2016-12-22 | Cepheid | Integrated purification and measurement of dna methylation and co-measurement of mutations and/or mrna expression levels in an automated reaction cartridge |
US10144724B2 (en) | 2015-07-22 | 2018-12-04 | Araxes Pharma Llc | Substituted quinazoline compounds and methods of use thereof |
CR20180164A (en) | 2015-08-18 | 2018-09-04 | Monsanto Technology Llc | METHODS TO PRODUCE COTTON PLANTS WITH IMPROVED DROUGHT TOLERANCE AND COMPOSITIONS OF THESE |
US10448595B2 (en) | 2015-09-03 | 2019-10-22 | Seminis Vegetable Seeds, Inc. | Downy mildew resistant lettuce plants |
WO2017044651A2 (en) | 2015-09-10 | 2017-03-16 | Beckman Coulter, Inc. | Short oligonucleotide quenchers for reduction of taqman probe baseline fluorescence |
US9499861B1 (en) | 2015-09-10 | 2016-11-22 | Insilixa, Inc. | Methods and systems for multiplex quantitative nucleic acid amplification |
WO2017044744A2 (en) | 2015-09-10 | 2017-03-16 | Monsanto Technology Llc | Methods for producing corn plants with downy mildew resistance and compositions thereof |
US10858343B2 (en) | 2015-09-28 | 2020-12-08 | Araxes Pharma Llc | Inhibitors of KRAS G12C mutant proteins |
US10875842B2 (en) | 2015-09-28 | 2020-12-29 | Araxes Pharma Llc | Inhibitors of KRAS G12C mutant proteins |
EP3356359B1 (en) | 2015-09-28 | 2021-10-20 | Araxes Pharma LLC | Inhibitors of kras g12c mutant proteins |
WO2017058915A1 (en) | 2015-09-28 | 2017-04-06 | Araxes Pharma Llc | Inhibitors of kras g12c mutant proteins |
WO2017058805A1 (en) | 2015-09-28 | 2017-04-06 | Araxes Pharma Llc | Inhibitors of kras g12c mutant proteins |
US10689356B2 (en) | 2015-09-28 | 2020-06-23 | Araxes Pharma Llc | Inhibitors of KRAS G12C mutant proteins |
EP3356351A1 (en) | 2015-09-28 | 2018-08-08 | Araxes Pharma LLC | Inhibitors of kras g12c mutant proteins |
EP3355914B1 (en) | 2015-09-29 | 2024-03-06 | The General Hospital Corporation | A composition comprising bcg for reducing cholesterol. |
EP3359689B1 (en) | 2015-10-08 | 2021-04-14 | Decipher Biosciences, Inc. | Use of a genetic signature diagnostically to evaluate treatment strategies for prostate cancer |
CN108699608A (en) | 2015-10-09 | 2018-10-23 | 基因前沿公司 | Method and kit for predicting and diagnosing human cytomegalovirus (hCMV) congenital transmission |
JP2018533939A (en) | 2015-10-19 | 2018-11-22 | アラクセス ファーマ エルエルシー | Method for screening for inhibitors of RAS |
WO2017070244A1 (en) | 2015-10-20 | 2017-04-27 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Biodosimetry analysis |
EP3371211A4 (en) | 2015-11-04 | 2019-08-21 | Icahn School of Medicine at Mount Sinai | Methods of treating tumors and cancer, and identifying candidate subjects for such treatment |
DK3168309T3 (en) | 2015-11-10 | 2020-06-22 | Eurofins Lifecodexx Gmbh | DETECTION OF Fetal Chromosomal Aneuploidies Using DNA Regions With Different Methylation Between Foster And The Pregnant Female |
AU2016355433C1 (en) | 2015-11-16 | 2021-12-16 | Araxes Pharma Llc | 2-substituted quinazoline compounds comprising a substituted heterocyclic group and methods of use thereof |
IL259466B (en) | 2015-11-20 | 2022-09-01 | Seegene Inc | Method for calibrating a data set of a target analyte |
US9988357B2 (en) | 2015-12-09 | 2018-06-05 | Araxes Pharma Llc | Methods for preparation of quinazoline derivatives |
KR102110985B1 (en) | 2015-12-15 | 2020-05-14 | 주식회사 씨젠 | Signal extraction for target nucleic acid sequences |
CA3002670A1 (en) | 2015-12-18 | 2017-06-22 | Donald Earl Kyle | Genetic loci associated with reproductive growth phenotypes in soybean and methods of use |
EP3389362A4 (en) | 2015-12-18 | 2019-08-07 | Monsanto Technology LLC | Methods for producing corn plants with northern leaf blight resistance and compositions thereof |
AU2017207341A1 (en) | 2016-01-12 | 2018-08-02 | Interleukin Genetics, Inc. | Methods for predicting response to treatment |
EP3405559B1 (en) | 2016-01-21 | 2021-11-17 | T2 Biosystems, Inc. | Rapid antimicrobial susceptibility testing using high-sensitivity direct detection methods |
EP3408403A1 (en) | 2016-01-25 | 2018-12-05 | Bio-Rad Europe GmbH | Digital microbiology |
JP6685138B2 (en) | 2016-01-27 | 2020-04-22 | シスメックス株式会社 | Quality control method for nucleic acid amplification, quality control reagent and reagent kit therefor |
EP3411819A4 (en) | 2016-02-05 | 2019-10-23 | Seegene, Inc. | Method for reducing noise level of data set for a target analyte |
CA3004914A1 (en) | 2016-02-05 | 2017-08-10 | Pioneer Hi-Bred International, Inc. | Genetic loci associated with brown stem rot resistance in soybean and methods of use |
EP3414344A1 (en) | 2016-02-08 | 2018-12-19 | The U.S.A. as represented by the Secretary, Department of Health and Human Services | Gene signature predictive of hepatocellular carcinoma response to transcatheter arterial chemoembolization (tace) |
WO2017155858A1 (en) | 2016-03-07 | 2017-09-14 | Insilixa, Inc. | Nucleic acid sequence identification using solid-phase cyclic single base extension |
WO2017172979A1 (en) | 2016-03-30 | 2017-10-05 | Araxes Pharma Llc | Substituted quinazoline compounds and methods of use |
WO2017173035A1 (en) | 2016-04-01 | 2017-10-05 | Chromacode Inc. | Competitive probes for engineering signal generation |
CN109072291A (en) | 2016-04-06 | 2018-12-21 | 生命技术公司 | For synthesizing and detecting composition, method and the kit of nucleic acid |
AU2017250205A1 (en) | 2016-04-14 | 2018-11-22 | T2 Biosystems, Inc. | Methods and systems for amplification in complex samples |
US11246868B2 (en) | 2016-04-26 | 2022-02-15 | Icahn School Of Medicine At Mount Sinai | Treatment of hippo pathway mutant tumors and methods of identifying subjects as candidates for treatment |
DE202017007130U1 (en) | 2016-04-27 | 2019-08-29 | Gen-Probe Inc. | Lysis reagent for blood cells |
US20200325234A1 (en) | 2016-04-27 | 2020-10-15 | Miradx | Immune-based treatment of kras-variant cancer patients |
JP7131773B2 (en) | 2016-04-29 | 2022-09-06 | ボード オブ リージェンツ,ザ ユニバーシティ オブ テキサス システム | A targeted measure of transcriptional activity associated with hormone receptors |
BR112018073236A2 (en) | 2016-05-12 | 2019-02-19 | Pioneer Hi Bred Int | methods for simultaneous clustered genotyping |
WO2017201302A1 (en) | 2016-05-18 | 2017-11-23 | The University Of Chicago | Btk mutation and ibrutinib resistance |
EP3469100B1 (en) | 2016-06-10 | 2021-01-27 | Seegene, Inc. | Methods for preparing tagging oligonucleotides |
CN109415772A (en) | 2016-06-16 | 2019-03-01 | 生物辐射实验室股份有限公司 | The method for detecting salmonella typhimurium |
WO2017218938A1 (en) | 2016-06-16 | 2017-12-21 | Life Technologies Corporation | Novel compositions, methods and kits for microorganism detection |
CN109642252A (en) | 2016-06-17 | 2019-04-16 | 加州理工学院 | Nucleic acid reaction and correlation technique and composition |
US11602752B2 (en) | 2016-06-30 | 2023-03-14 | Seegene, Inc. | Apparatus for amplificating nucleic acid and fluorescence-detecting device |
US10646488B2 (en) | 2016-07-13 | 2020-05-12 | Araxes Pharma Llc | Conjugates of cereblon binding compounds and G12C mutant KRAS, HRAS or NRAS protein modulating compounds and methods of use thereof |
US11421287B2 (en) | 2016-07-29 | 2022-08-23 | Juno Therapeutics, Inc. | Methods for assessing the presence or absence of replication competent virus |
US10837067B2 (en) | 2016-08-11 | 2020-11-17 | Monsanto Technology Llc | Methods and compositions for producing corn plants with resistance to late wilt |
AU2017315425B2 (en) | 2016-08-24 | 2023-11-09 | The Regents Of The University Of Michigan | Use of genomic signatures to predict responsiveness of patients with prostate cancer to post-operative radiation therapy |
EP3504342A1 (en) | 2016-08-26 | 2019-07-03 | Life Technologies Corporation | Nucleic acid extraction and amplification controls and methods of use thereof |
WO2018045162A1 (en) | 2016-09-01 | 2018-03-08 | Biogen Ma Inc. | Biomarkers predictive of primary progressive multiple sclerosis and uses thereof |
ES2893411T3 (en) | 2016-09-15 | 2022-02-09 | Hoffmann La Roche | Procedures for performing multiplexed PCR |
JP2019529484A (en) | 2016-09-29 | 2019-10-17 | アラクセス ファーマ エルエルシー | Inhibitor of KRAS G12C mutant protein |
AU2017232187B2 (en) | 2016-09-30 | 2023-11-09 | Seminis Vegetable Seeds, Inc. | Xanthomonas resistant brassica oleracea plants |
US10377743B2 (en) | 2016-10-07 | 2019-08-13 | Araxes Pharma Llc | Inhibitors of RAS and methods of use thereof |
WO2018089953A1 (en) | 2016-11-14 | 2018-05-17 | Orca Biosystems, Inc. | Methods and apparatuses for sorting target particles |
US11260387B2 (en) | 2016-12-12 | 2022-03-01 | Cepheid | Integrated purification and measurement of DNA methylation and co-measurement of mutations and/or mRNA expression levels in an automated reaction cartridge |
EP3551766B1 (en) | 2016-12-12 | 2022-09-14 | Cepheid | Integrated immuno-pcr and nucleic acid analysis in an automated reaction cartridge |
EP3555077B1 (en) | 2016-12-15 | 2023-04-19 | The Regents of The University of California | Compositions and methods for treating cancer |
US10427162B2 (en) | 2016-12-21 | 2019-10-01 | Quandx Inc. | Systems and methods for molecular diagnostics |
EP4001428B1 (en) | 2016-12-29 | 2023-11-22 | Seegene, Inc. | Method for reducing primer dimer formation and increasing amplification efficiency |
US10329620B2 (en) | 2017-01-12 | 2019-06-25 | Cardioforecast Ltd. | Methods and kits for treating cardiovascular disease |
WO2018132916A1 (en) | 2017-01-20 | 2018-07-26 | Genomedx Biosciences, Inc. | Molecular subtyping, prognosis, and treatment of bladder cancer |
EP3573971A1 (en) | 2017-01-26 | 2019-12-04 | Araxes Pharma LLC | 1-(3-(6-(3-hydroxynaphthalen-1-yl)benzofuran-2-yl)azetidin-1yl)prop-2-en-1-one derivatives and similar compounds as kras g12c modulators for treating cancer |
US11358959B2 (en) | 2017-01-26 | 2022-06-14 | Araxes Pharma Llc | Benzothiophene and benzothiazole compounds and methods of use thereof |
EP3573967A1 (en) | 2017-01-26 | 2019-12-04 | Araxes Pharma LLC | Fused hetero-hetero bicyclic compounds and methods of use thereof |
EP3573954A1 (en) | 2017-01-26 | 2019-12-04 | Araxes Pharma LLC | Fused bicyclic benzoheteroaromatic compounds and methods of use thereof |
US11136308B2 (en) | 2017-01-26 | 2021-10-05 | Araxes Pharma Llc | Substituted quinazoline and quinazolinone compounds and methods of use thereof |
EP3592860B1 (en) | 2017-03-08 | 2021-11-03 | Etablissement Français du Sang | Rhd gene allele associated with a weak d phenotype and its uses |
CA3055925A1 (en) | 2017-03-09 | 2018-09-13 | Decipher Biosciences, Inc. | Subtyping prostate cancer to predict response to hormone therapy |
US20180265887A1 (en) | 2017-03-16 | 2018-09-20 | Jacobs Farm Del Cabo | Basil Plants With High Tolerance to Downy Mildew |
CA3176529C (en) | 2017-03-24 | 2024-03-05 | Gen-Probe Incorporated | Compositions and methods for detection of viral pathogens in samples |
US11078542B2 (en) | 2017-05-12 | 2021-08-03 | Decipher Biosciences, Inc. | Genetic signatures to predict prostate cancer metastasis and identify tumor aggressiveness |
US20190093155A1 (en) * | 2017-05-25 | 2019-03-28 | Roche Molecular Systems, Inc. | Multiplex Nucleic Acid Amplification Assay |
KR20200010306A (en) | 2017-05-25 | 2020-01-30 | 아락세스 파마 엘엘씨 | Covalent Inhibitors of KRAS |
WO2018218069A1 (en) | 2017-05-25 | 2018-11-29 | Araxes Pharma Llc | Quinazoline derivatives as modulators of mutant kras, hras or nras |
US10736897B2 (en) | 2017-05-25 | 2020-08-11 | Araxes Pharma Llc | Compounds and methods of use thereof for treatment of cancer |
US11217329B1 (en) | 2017-06-23 | 2022-01-04 | Veracyte, Inc. | Methods and systems for determining biological sample integrity |
US20220225597A1 (en) | 2017-06-29 | 2022-07-21 | Juno Therapeutics, Inc. | Mouse model for assessing toxicities associated with immunotherapies |
EP4289507A3 (en) | 2017-07-10 | 2024-02-28 | Gen-Probe Incorporated | Analytical systems and methods for nucleic acid amplification using sample assigning parameters |
KR20200064060A (en) | 2017-07-29 | 2020-06-05 | 주노 쎄러퓨티크스 인코퍼레이티드 | Reagent for cell amplification expressing a recombinant receptor |
KR102380264B1 (en) | 2017-08-31 | 2022-03-29 | 주식회사 씨젠 | Evaluation of the performance of components using dimer-forming primer pairs |
WO2019046832A1 (en) | 2017-09-01 | 2019-03-07 | Juno Therapeutics, Inc. | Gene expression and assessment of risk of developing toxicity following cell therapy |
KR102345601B1 (en) | 2017-09-29 | 2021-12-30 | 주식회사 씨젠 | Detection of target nucleic acid sequences by PTO cleavage and extension-dependent extension analysis |
US11060133B2 (en) | 2017-10-26 | 2021-07-13 | Arizona Board Of Regents On Behalf Of Arizona State University | Methods for detection and quantification of infectious carbapenem resistant Enterobacteriaceae (CRE) |
CA3080509A1 (en) | 2017-11-01 | 2019-05-09 | Juno Therapeutics, Inc. | Process for producing a t cell composition |
CN111527216A (en) | 2017-11-13 | 2020-08-11 | 生命技术公司 | Compositions, methods and kits for urinary tract microbial detection |
WO2019103967A1 (en) | 2017-11-22 | 2019-05-31 | The Regents Of The University Of Michigan | Compositions and methods for treating cancer |
EP3735476B1 (en) | 2018-01-05 | 2023-08-23 | Seegene, Inc. | Method for determining the presence or absence of m. tuberculosis, m. bovis and m. bovis bcg in a sample |
CN111902213B (en) | 2018-01-29 | 2022-12-06 | 简·探针公司 | Analysis system and method |
US11535903B2 (en) | 2018-01-31 | 2022-12-27 | Juno Therapeutics, Inc. | Methods and reagents for assessing the presence or absence of replication competent virus |
JP7170062B2 (en) | 2018-04-20 | 2022-11-11 | シージーン アイエヌシー | Methods and apparatus for detection of multiple target nucleic acid sequences within a sample |
US20210239700A1 (en) | 2018-05-04 | 2021-08-05 | Abbott Laboratories | Hbv diagnostic, prognostic, and therapeutic methods and products |
US11268102B2 (en) | 2018-05-16 | 2022-03-08 | University Of Florida Research Foundation, Incorporated | Compositions and methods for identifying and selecting brachytic locus in solanaceae |
JP7470095B2 (en) | 2018-07-10 | 2024-04-17 | ジェン-プローブ・インコーポレーテッド | Methods and systems for detecting and quantifying nucleic acids - Patents.com |
WO2020014296A1 (en) | 2018-07-12 | 2020-01-16 | Luminex Corporation | Systems and methods for performing variable sample preparation and analysis processes |
GB201812192D0 (en) | 2018-07-26 | 2018-09-12 | Ttp Plc | Variable temperature reactor, heater and control circuit for the same |
AU2019319901B2 (en) | 2018-08-10 | 2022-07-07 | Life Technologies Corporation | Silicon-substituted rhodamine dyes and dye conjugates |
WO2020047081A1 (en) | 2018-08-30 | 2020-03-05 | Life Technologies Corporation | Machine learning system for genotyping pcr assays |
WO2020047288A1 (en) | 2018-08-31 | 2020-03-05 | Life Technologies Corporation | Image driven quality control for array based pcr |
US20210324452A1 (en) | 2018-09-06 | 2021-10-21 | Hygiena, Llc | Sequences and their use for detection and characterization of escherichia coli serotype o157:h7 |
CA3112792A1 (en) | 2018-09-14 | 2020-03-19 | Prelude Corporation | Method of selection for treatment of subjects at risk of invasive breast cancer |
CA3112342A1 (en) | 2018-10-01 | 2020-04-09 | Gen-Probe Incorporated | Compositions and methods for amplifying or detecting varicella-zoster virus |
CN111004865A (en) | 2018-10-05 | 2020-04-14 | 福又达生物科技股份有限公司 | Method for detecting fish pathogens |
WO2020097132A1 (en) | 2018-11-06 | 2020-05-14 | Juno Therapeutics, Inc. | Process for producing genetically engineered t cells |
EP3899035A1 (en) | 2018-12-20 | 2021-10-27 | F. Hoffmann-La Roche AG | Detection of target nucleic acid by solid-phase molography |
WO2020150144A1 (en) | 2019-01-15 | 2020-07-23 | Seminis Vegetable Seeds, Inc. | Green bean plants with improved disease resistance |
KR102097721B1 (en) | 2019-01-24 | 2020-04-06 | 주식회사 시선바이오머티리얼스 | Method for Detecting Multiple Target Based on Single Detection Probe using Tag sequence SNP |
EP3937780A4 (en) | 2019-03-14 | 2022-12-07 | InSilixa, Inc. | Methods and systems for time-gated fluorescent-based detection |
US11634782B2 (en) | 2019-03-20 | 2023-04-25 | Hygiena, Llc | Quantification of microorganisms in samples and methods of determining quantification conditions thereof |
EP3719144A1 (en) | 2019-04-05 | 2020-10-07 | Fundación para la Investigación Biomédica del Hospital Universitario de la Paz (FIBHULP) | Mir-151a-3p as an universal endogenous control for exosome cargo normalization |
WO2020205491A1 (en) | 2019-04-05 | 2020-10-08 | Hygiena, Llc | Sequences and their use for detection and characterization of genus cronobacter |
MX2021012607A (en) | 2019-04-17 | 2022-03-11 | Alpine Immune Sciences Inc | Methods and uses of variant icos ligand (icosl) fusion proteins. |
US20220349910A1 (en) | 2019-05-03 | 2022-11-03 | Gen-Probe Incorporated | Receptacle delivery system |
EP3980561A1 (en) | 2019-06-06 | 2022-04-13 | Sitokine Limited | Compositions and methods for treating lung, colorectal and breast cancer |
CA3142361A1 (en) | 2019-06-12 | 2020-12-17 | Juno Therapeutics, Inc. | Combination therapy of a cell-mediated cytotoxic therapy and an inhibitor of a prosurvival bcl2 family protein |
CA3138015A1 (en) | 2019-06-14 | 2020-12-17 | Jong Yoon Chun | Computer-implemented method for collaborative development of reagents for detection of target nucleic acids |
RU2723390C1 (en) * | 2019-07-04 | 2020-06-11 | Объединенный Институт Ядерных Исследований (Оияи) | Method for prediction of risk of developing diseases associated with level of immunoglobulin e (ige) in human blood serum |
US20220267857A1 (en) | 2019-07-19 | 2022-08-25 | Fundación Para La Investigación Biomédikca Del Hospital Universitario La Paz (Fibhulp) | Method for determining the response to treatment of a patient affected by non-small cell lung carcinoma (nsclc) |
WO2021028469A1 (en) | 2019-08-12 | 2021-02-18 | Sitokine Limited | Compositions and methods for treating cytokine release syndrome and neurotoxicity |
KR20220066892A (en) | 2019-08-22 | 2022-05-24 | 주노 쎄러퓨티크스 인코퍼레이티드 | Combination therapy of T cell therapy and Zest homologue 2 enhancer (EH2) inhibitor and related methods |
SG10202008262UA (en) | 2019-09-26 | 2021-04-29 | Seminis Vegetable Seeds Inc | Lettuce plants having resistance to nasonovia ribisnigri biotype nr:1 |
WO2021140173A1 (en) | 2020-01-10 | 2021-07-15 | Biouniversa S.R.L. | Methods and uses for treating fibrotic solid tumors with bags inhibitors |
MX2022008625A (en) | 2020-01-13 | 2022-09-09 | Aspen Neuroscience Inc | Method of differentiating neural cells and related compositions and methods of use. |
FI130554B (en) | 2020-01-20 | 2023-11-16 | Oy Arctic Partners Ab | Luminescence hybridisation assay method |
AU2021224298A1 (en) | 2020-02-18 | 2022-09-22 | Life Technologies Corporation | Compositions, kits and methods for detection of viral sequences |
WO2021205013A1 (en) | 2020-04-09 | 2021-10-14 | Sitokine Limited | Compositions and methods for treating covid-19 |
WO2021231661A2 (en) | 2020-05-13 | 2021-11-18 | Juno Therapeutics, Inc. | Process for producing donor-batched cells expressing a recombinant receptor |
WO2021236651A1 (en) * | 2020-05-19 | 2021-11-25 | The Regents Of The University Of California | Compositions and methods of a nuclease chain reaction for nucleic acid detection |
WO2021246820A1 (en) | 2020-06-05 | 2021-12-09 | 주식회사 씨젠 | Specimen transport kit for detecting respiratory pathogens and methods for detecting respiratory pathogens using same |
CN116234558A (en) | 2020-06-26 | 2023-06-06 | 朱诺治疗学有限公司 | Engineered T cells conditionally expressing recombinant receptors, related polynucleotides and methods |
AU2021204717A1 (en) | 2020-07-15 | 2022-02-03 | Seminis Vegetable Seeds, Inc. | Green Bean Plants with Improved Disease Resistance |
CA3186950A1 (en) | 2020-07-23 | 2022-01-27 | Scott Benson | Compositions, systems and methods for biological analysis involving energy transfer dye conjugates and analytes comprising the same |
CA3186955A1 (en) | 2020-07-23 | 2022-01-27 | Scott Benson | Energy transfer dye conjugates for use in biological assays |
RU2741838C1 (en) * | 2020-08-19 | 2021-01-29 | Объединенный Институт Ядерных Исследований (Оияи) | Method for determining the presence of a genetic predisposition to human longevity |
WO2022087257A2 (en) | 2020-10-21 | 2022-04-28 | Gen-Probe Incorporated | Fluid container management system |
GB2616359A (en) | 2020-11-20 | 2023-09-06 | Decipher Biosciences Inc | Methods and genomic classifiers for identifying homologous recombination deficiency prostate cancer |
JP2023551542A (en) | 2020-11-30 | 2023-12-08 | エニグマ バイオインテリジェンス,インコーポレイテッド | Non-invasive assessment of Alzheimer's disease |
JP7209980B2 (en) | 2020-12-11 | 2023-01-23 | 東洋紡株式会社 | An antibody that specifically binds to the 5' to 3' exonuclease active domain of a DNA polymerase |
AU2021410073A1 (en) | 2020-12-21 | 2023-07-06 | BASF Agricultural Solutions Seed US LLC | Brassica napus plants comprising an improved fertility restorer |
CN116601308A (en) | 2020-12-22 | 2023-08-15 | 豪夫迈·罗氏有限公司 | Method for multiplex real-time PCR using large Stokes shift fluorescent dyes |
EP4278002A2 (en) | 2021-01-18 | 2023-11-22 | Life Technologies Corporation | Compositions, kits and methods for direct amplification from crude biological samples |
EP4281589A1 (en) | 2021-01-25 | 2023-11-29 | Life Technologies Corporation | Compositions, kits and methods for detection of viral variant sequences |
WO2022178216A2 (en) | 2021-02-22 | 2022-08-25 | Cotropia Joseph | Corona virus antigens and epitopes and proteins that bind thereto |
EP4308588A1 (en) | 2021-03-18 | 2024-01-24 | Cotropia, Joseph | Human immunodeficiency virus (hiv) antigens and epitopes and proteins that bind thereto |
EP4314350A1 (en) | 2021-03-23 | 2024-02-07 | Life Technologies Corporation | Compositions, kits, and methods for variant-resistant detection of target viral sequences |
IL307530A (en) | 2021-04-06 | 2023-12-01 | Bpgbio Inc | Protein markers for estrogen receptor (er)-positive luminal a(la)-like and luminal b1 (lb1)-like breast cancer |
IL307528A (en) | 2021-04-06 | 2023-12-01 | Bpgbio Inc | Protein markers for the prognosis of breast cancer progression |
WO2022216846A1 (en) | 2021-04-06 | 2022-10-13 | Berg Llc | Protein markers for estrogen receptor (er)-positive-like and estrogen receptor (er)-negative-like breast cancer |
CA3217862A1 (en) | 2021-05-05 | 2022-11-10 | Radius Pharmaceuticals, Inc. | Animal model having homologous recombination of mouse pth1 receptor |
WO2022269023A1 (en) | 2021-06-25 | 2022-12-29 | F. Hoffmann-La Roche Ag | Methods for performing temperature multiplexed pcr with increased sensitivity |
CN117897454A (en) | 2021-07-21 | 2024-04-16 | 生命技术公司 | Dibenzoxanthene quencher, use and preparation method |
WO2023004356A2 (en) | 2021-07-21 | 2023-01-26 | Life Technologies Corporation | Azaindole cyanine dyes, uses, and methods of preparation |
WO2023004371A1 (en) | 2021-07-21 | 2023-01-26 | Aspen Neuroscience, Inc. | Methods of differentiating neural cells and predicting engraftment thereof and related compositions |
CN117794357A (en) | 2021-07-23 | 2024-03-29 | 巴斯夫农业种子解决方案美国有限责任公司 | Black shank-resistant plants and methods for identifying black shank-resistant plants |
WO2023006642A1 (en) | 2021-07-26 | 2023-02-02 | Glaxosmithkline Intellectual Property Development Limited | Assay methods for titration of retrovirus and retroviral vectors |
FR3125824A1 (en) | 2021-07-30 | 2023-02-03 | Bforcure | Device and method for multiplexed detection of nucleic acid sequences |
WO2023014729A1 (en) | 2021-08-02 | 2023-02-09 | Life Technologies Corporation | Compositions, kits, and methods for detection of nucleic acid sequence loads |
WO2023031770A1 (en) | 2021-08-30 | 2023-03-09 | Galderma Holding SA | Treatments for atopic dermatitis |
WO2023039525A1 (en) | 2021-09-10 | 2023-03-16 | Life Technologies Corporation | Master mix compositions, kits, and methods |
WO2023043280A1 (en) | 2021-09-17 | 2023-03-23 | 주식회사 씨젠 | Detection of target nucleic acid sequence by using synthetic non-natural base-bearing tag oligonucleotide |
WO2023069604A1 (en) | 2021-10-20 | 2023-04-27 | Life Technologies Corporation | Compositions, kits, and methods for quantification of nucleic acid sequences using an internal quantitative standard |
CA3229091A1 (en) | 2021-10-26 | 2023-05-04 | Caribou Biosciences, Inc. | Exonuclease-coupled real-time endonuclease activity assay |
WO2023081221A1 (en) | 2021-11-02 | 2023-05-11 | Life Technologies Corporation | Compositions and methods for reducing master mix contamination |
WO2023102459A1 (en) | 2021-12-03 | 2023-06-08 | Medicinal Genomics Corporation | Psilocybe assay |
US20230193310A1 (en) | 2021-12-10 | 2023-06-22 | Seminis Vegetabe Seeds, Inc. | Lettuce plants having resistance to downy mildew |
WO2023201361A1 (en) | 2022-04-15 | 2023-10-19 | Aspen Neuroscience, Inc. | Methods of classifying the differentiation state of cells and related compositions of differentiated cells |
WO2023230581A1 (en) | 2022-05-25 | 2023-11-30 | Celgene Corporation | Methods of manufacturing t cell therapies |
US20230404003A1 (en) | 2022-06-21 | 2023-12-21 | Seminis Vegetable Seeds, Inc. | Novel qtls conferring resistance to cucumber mosaic virus |
WO2024002924A2 (en) | 2022-06-28 | 2024-01-04 | F. Hoffmann-La Roche Ag | Fluorescent dyes with large stokes shift |
WO2024006927A1 (en) | 2022-06-29 | 2024-01-04 | Life Technologies Corporation | Compositions, kits, and methods for detecting nucleic acids using intra-channel multiplexing |
WO2024006924A1 (en) | 2022-06-29 | 2024-01-04 | Life Technologies Corporation | Systems and methods for analyte detection from multiplexed assays |
WO2024044352A1 (en) | 2022-08-26 | 2024-02-29 | The General Hospital Corporation | Methods and compositions for prognosis and treatment of dilated cardiomyopathy and heart failure |
WO2024054925A1 (en) | 2022-09-09 | 2024-03-14 | Life Technologies Corporation | Compositions, kits and methods for detection of viral variant sequences |
WO2024059493A1 (en) | 2022-09-13 | 2024-03-21 | Medicinal Genomics Corporation | Psilocybe assay |
Family Cites Families (24)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4220450A (en) * | 1978-04-05 | 1980-09-02 | Syva Company | Chemically induced fluorescence immunoassay |
JPS613063A (en) * | 1984-06-18 | 1986-01-09 | Fujirebio Inc | Measurement of polynucleotide utilizing luminous substance |
US4996143A (en) | 1985-12-23 | 1991-02-26 | Syngene, Inc. | Fluorescent stokes shift probes for polynucleotide hybridization |
CA1273552A (en) * | 1985-12-23 | 1990-09-04 | Michael J. Heller | Fluorescent stokes shift probes for polynucleotide hybridization assays |
ATE88761T1 (en) * | 1986-01-10 | 1993-05-15 | Amoco Corp | COMPETITIVE HOMOGENEOUS TEST. |
EP0343955A3 (en) * | 1988-05-27 | 1990-04-18 | Bioventures, Inc. | Process for complex binding of targeted molecular species |
US5118801A (en) * | 1988-09-30 | 1992-06-02 | The Public Health Research Institute | Nucleic acid process containing improved molecular switch |
AU6309690A (en) | 1989-09-28 | 1991-04-11 | Abbott Laboratories | Stabilization of monoclonal antibody for use in fluorescent polarization techniques |
DE69126915T2 (en) | 1990-05-16 | 1998-02-19 | Abbott Lab | Test for barbiturates, tracers, immunogens, antibodies and test kit therefor |
US5210015A (en) | 1990-08-06 | 1993-05-11 | Hoffman-La Roche Inc. | Homogeneous assay system using the nuclease activity of a nucleic acid polymerase |
DE4123540A1 (en) * | 1991-07-16 | 1993-01-21 | Boehringer Mannheim Gmbh | IMMOBILIZATION OF NUCLEIC ACIDS |
JP3085756B2 (en) * | 1991-10-30 | 2000-09-11 | 株式会社日立製作所 | Nucleic acid detection method |
US5747244A (en) * | 1991-12-23 | 1998-05-05 | Chiron Corporation | Nucleic acid probes immobilized on polystyrene surfaces |
US5332659A (en) * | 1992-04-09 | 1994-07-26 | The United States Of America As Represented By The Secretary Of The Navy | Light emission-or absorbance-based binding assays for polynucleic acids |
EP0601889A2 (en) * | 1992-12-10 | 1994-06-15 | Maine Medical Center Research Institute | Nucleic acid probes |
US6482655B1 (en) * | 1993-07-23 | 2002-11-19 | University Of Utah Research Foundation | Immunoassay procedure utilizing fluorogenic tracer antigens |
US5925517A (en) | 1993-11-12 | 1999-07-20 | The Public Health Research Institute Of The City Of New York, Inc. | Detectably labeled dual conformation oligonucleotide probes, assays and kits |
US5538848A (en) | 1994-11-16 | 1996-07-23 | Applied Biosystems Division, Perkin-Elmer Corp. | Method for detecting nucleic acid amplification using self-quenching fluorescence probe |
DE4442011C2 (en) * | 1993-11-29 | 1997-09-11 | Gerhard Ing Grad Hiesl | Method and device for filling the recesses of an object with a heat-liquid material, in particular the recesses of plain bearing bushes with wax |
US5654419A (en) | 1994-02-01 | 1997-08-05 | The Regents Of The University Of California | Fluorescent labels and their use in separations |
US5565554A (en) | 1994-07-29 | 1996-10-15 | The Regents Of The University Of California | Dimeric fluorescent energy transfer dyes comprising asymmetric cyanine azole-indolenine chromophores |
US5491063A (en) * | 1994-09-01 | 1996-02-13 | Hoffmann-La Roche Inc. | Methods for in-solution quenching of fluorescently labeled oligonucleotide probes |
US5741657A (en) | 1995-03-20 | 1998-04-21 | The Regents Of The University Of California | Fluorogenic substrates for β-lactamase and methods of use |
US6008373A (en) | 1995-06-07 | 1999-12-28 | Carnegie Mellon University | Fluorescent labeling complexes with large stokes shift formed by coupling together cyanine and other fluorochromes capable of resonance energy transfer |
-
1994
- 1994-11-16 US US08/340,558 patent/US5538848A/en not_active Expired - Lifetime
-
1995
- 1995-11-15 JP JP51632196A patent/JP4131749B2/en not_active Expired - Lifetime
- 1995-11-15 EP EP99201972A patent/EP0972848A3/en not_active Withdrawn
- 1995-11-15 CA CA002201756A patent/CA2201756C/en not_active Expired - Fee Related
- 1995-11-15 WO PCT/US1995/014882 patent/WO1996015270A1/en active IP Right Grant
- 1995-11-15 US US08/559,405 patent/US5723591A/en not_active Expired - Lifetime
- 1995-11-15 DE DE69519940T patent/DE69519940T2/en not_active Expired - Lifetime
- 1995-11-15 US US08/558,303 patent/US5876930A/en not_active Expired - Lifetime
- 1995-11-15 AT AT95941402T patent/ATE198775T1/en active
- 1995-11-15 EP EP95941402A patent/EP0792374B1/en not_active Expired - Lifetime
-
1998
- 1998-12-07 US US09/207,170 patent/US6030787A/en not_active Expired - Lifetime
-
1999
- 1999-11-08 US US09/436,454 patent/US6258569B1/en not_active Expired - Lifetime
-
2002
- 2002-09-05 JP JP2002260129A patent/JP2003144198A/en active Pending
-
2005
- 2005-03-16 JP JP2005075576A patent/JP2005176858A/en active Pending
Also Published As
Publication number | Publication date |
---|---|
WO1996015270A1 (en) | 1996-05-23 |
DE69519940T2 (en) | 2001-05-23 |
US5723591A (en) | 1998-03-03 |
JP2005176858A (en) | 2005-07-07 |
DE69519940D1 (en) | 2001-02-22 |
EP0792374B1 (en) | 2001-01-17 |
JP4131749B2 (en) | 2008-08-13 |
US5538848A (en) | 1996-07-23 |
EP0792374A1 (en) | 1997-09-03 |
EP0972848A3 (en) | 2000-10-04 |
US5876930A (en) | 1999-03-02 |
AU695561B2 (en) | 1998-08-13 |
ATE198775T1 (en) | 2001-02-15 |
EP0972848A2 (en) | 2000-01-19 |
JPH10510982A (en) | 1998-10-27 |
AU4283696A (en) | 1996-06-06 |
US6258569B1 (en) | 2001-07-10 |
JP2003144198A (en) | 2003-05-20 |
CA2201756A1 (en) | 1996-05-23 |
US6030787A (en) | 2000-02-29 |
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