WO1997046703A1 - Optimally fluorescent oligonucleotides - Google Patents
Optimally fluorescent oligonucleotides Download PDFInfo
- Publication number
- WO1997046703A1 WO1997046703A1 PCT/US1997/009270 US9709270W WO9746703A1 WO 1997046703 A1 WO1997046703 A1 WO 1997046703A1 US 9709270 W US9709270 W US 9709270W WO 9746703 A1 WO9746703 A1 WO 9746703A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- nucleotide
- oligonucleotide
- sequence
- conjugated
- template
- Prior art date
Links
Classifications
-
- 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
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- 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
- C12Q1/682—Signal amplification
-
- 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
- C12Q1/6841—In situ hybridisation
Definitions
- This invention relates to the preparation and use of labeled oligonucleotides. More particularly, it relates to methods of preparing and using fluorescently labeled nucleotides.
- fluorescent labels of nucleic acids can be divided into two classes: (1) those which covalently modify nucleic acids with a fluorescent moiety, and (2) those which non-covalently modify nucleic acids with a fluorescent moiety, i.e., by ionic interactions, hydrogen-bonding, or intercalation.
- non-covalent fluorescent probes of nucleic acids exhibit dramatically increased fluorescence upon binding to nucleic acids, and consequently, have been very useful in assays designed to determine the total nucleic acid present in a given sample.
- non-covalently bound fluorescent molecules can, and will, migrate from a labeled strand to an unlabeled one.
- Covalently bound fluorescent molecules on the other hand, can not migrate from a labeled oligonucleotide to an unlabeled one. Therefore covalently bound fluorescent moieties are preferred for use as fluorescently tagged nucleic acid probes.
- Examples of fluorescent compounds which have been covalently attached to nucleic acid sequences include conjugates between nucleotide triphosphates or phosphoramidites and fluorescent moieties, and directly reactive dyes.
- Nucleotide triphosphates are incorporated into a nucleic acids by nucleic acid polymerases.
- Commercially available nucleotide triphosphates-dye conjugates include dCTP-Cy3, dCTP-Cy5, dUTP-FluorX, etc. available from DuPont, Molecular Probes, Boehringer Mannheim, and Amersham Life Sciences.
- dye conjugates contain cyanine or fluorescein derivatives which are covalently bound to the nucleotide, and each dye conjugate differs with respect to the absorbance maxima of the dye moiety.
- Directly reactive dyes covalently bind to an existing nucleic acid sequence.
- a few reactive dyes are commercially available, including various psoralens and ethidium mono- and di-azides.
- Fluorescently labeled oligonucleotides have also been synthesized by a combination of modified phosphoramidites and reactive dyes, typically involving the incorporation of primary amines in the oligonucleotide during synthesis followed by covalent coupling of the amine groups to a reactive dye.
- nucleotide triphosphate-dye conjugates offer the greatest flexibility and the highest achievable specific fluorescence.
- Synthetic nucleic acids molecules produced non-enzymatically
- Directly reactive dyes such as ethidium monoazide, react non-specifically and can potentially damage the labeled oligonucleotide.
- Polymerase-driven labeling can produce molecules from a few tens of bases to several kilobases, can utilize standard labeling methods such as nick translation and primer extension reactions, and the degree of dye incorporation can be roughly controlled by varying the ratio of labeled NTP to unlabeled NTP.
- the primary limitation of polymerase-driven fluorescent labeling of nucleic acids is the absence of absolute control of the amount of fluorescent compound incorporated into a particular sequence. For example, if one desires to label DNA with dCTP-Cy3 and the specific sequence has only a limited number of "C" sites, then the resulting fluorescently labeled oligonucleotide will have relatively few Cy3 molecules and consequently a low specific fluorescence.
- the present invention overcomes this sequence specific limitation and optimizes the incorporation of the fluorescent moiety by polyraerase.
- the invention provides a method for the preparation and purification of optimally fluorescent oligonucleotides comprising the steps of (a) preparing a primer; (b) preparing a template oligonucleotide containing a nucleotide sequence complementary to the primer, and a nucleotide repeat region downstream from the the complementary region; (c) annealing the template and primer in a suitable reaction medium containing a polymerase, nucleotide triphosphates and fluorescent dye- conjugated nucleotide triphosphates; (d) initiating synthesis of a complementary strand on the template; (e) attaching the oligonucleotide containing a target sequence adjacent to the complementary strand; and (f) purifying the optimally fluorescent oligonucleotide by any appropriate method.
- the invention also provides an oligonucleotide comprising a nucleotide sequence complementary to a primer, and a nucleotide repeat region downstream from said complementary sequence, wherein the nucleotide repeat region comprises N* 1 where N fc is any nucleotide which can form a base pair with a fluorescent dye- conjugated nucleotide triphosphate, and optionally, a plurality of nucleotides, N, which are not capable of forming a base pair with a fluorescent dye-conjugated nucleotide triphosphate.
- the invention further provides an optimally fluorescent comprising a radiolabeled nucleic acid sequence and a nucleotide repeat region, wherein the nucleotide repeat region comprises N f , where N f is any nucleotide which is conjugated to a fluorescent dye, and optimally, a plurality of nucleotides, N, which are not conjugated to a fluorescent dye.
- the "complement" to a first nucleotide sequence is well known to be a second sequence comprising those bases which will pair by Watson-Crick hybridization with the first sequence.
- the complement to the deoxyribonucleic acid (DNA) sequence 5'-ATGC 3' is well known to be 5'-GCAT 3'.
- DNA deoxyribonucleic acid
- 5'-ATGC 3' is well known to be 5'-GCAT 3'.
- each of the two strands are described as complementary to the other or as a complementary pair.
- complement and anticomplement may also be used.
- the transcription strand is generally described as plus and its complement as minus (or "+" and •'-") , or the transcription strand may be described as the sense strand, and its complement as antisense.
- Two strands each hybridized to the other having all base pairs complementary, are 100% complementary to each other.
- Two strands, each hybridized to the other, having 5% of bases non-complementary, are 95% complementary (or the two strands have 95% complementarity) .
- a “probe” is a single or double stranded nucleic acid which has a sequence complementary to a target nucleic acid sequence of interest and which has some additional feature enabling the measurement of the probe-target duplex.
- the artisan will understand that if the probe and/or the target is double stranded, the double stranded nucleic acid must undergo strand separation before hybridization can take place.
- a probe is rendered detectable by an attached tag or label.
- a tag or label linked to a probe may include, in principle, a fluorescent or luminescent tag, an isotopic label, a dye label, an enzyme label, an antigen determinant detectable by an antibody, or a binding moiety such as biotin enabling yet another moiety such as a streptavidin coated bead to specifically attach the probe.
- a binding moiety such as biotin enabling yet another moiety such as a streptavidin coated bead to specifically attach the probe.
- a “primer” is a relatively short segment of oligonucleotide which is complementary to a portion of the sequence of interest (the sequence of interest can be a subfragment within a larger nucleic acid sequence) .
- a primer represents the 5' terminus of the resulting extension product.
- a primer which is complementary to the sequence of interest on the template strand enables the 3' terminus to be acted on by a polymerase.
- a primer may also be modified at its 5' end with a binding moiety or detectable label.
- Hybridization describes the formation of double stranded or duplex nucleic acid from complementary single stranded nucleic acids. Hybridization may take place between sufficiently complementary single stranded DNA and/or RNA to form: DNA-DNA, DNA-RNA or RNA-RNA.
- DNA polymerase The in vitro amplification of DNA is catalyzed by DNA polymerase.
- a number of types of DNA polymerase are known in the art. They generally share the common property of catalyzing the synthesis of a double stranded DNA sequence utilizing a single stranded template to which a primer is annealed.
- DNA polymerases extracted from most organisms become inactive at the temperatures required for thermal denaturing of nucleic acids. Thus, replacement of the enzyme at the start of each thermal cycle, or the addition of a factor able to prevent heat inactivation, is required if such heat sensitive enzymes are utilized.
- the DNA polymerases which are preferred for in vitro PCR as well as for the invention are derived from organisms which thrive at high temperatures and thus are heat resistant, i.e., thus maintain adequate catalytic activity at the temperature which denatures duplex DNA.
- the reaction catalyzed by DNA polymerase is known to the art, and referred to herein as the "DNA polymerase reaction".
- the reaction requires some or all of the four deoxyribonucleotide triphosphates and primers, preferably in molar excess, and a means for cyclic strand separation. Strand separation is preferably achieved by thermal cycling between annealing and denaturation temperatures.
- Reverse transcriptase is known to mediate both RNA and DNA copying, as well as DNA to DNA copying. Hence, any number of enzymes now known will catalyze the polymerization reaction.
- Optimal spacing describes that distance between fluorescently labeled nucleotides which results in the maximum fluorescence of the oligonucleotide.
- Specific fluorescence refers to the quantum efficiency per unit mass of labeled nucleic acid, or the amount of fluorescent label incorporated per unit mass of labeled nucleic acid.
- Optimal fluorescence refers to the maximum specific fluorescence which can be obtained in a given reaction medium, and it is based on the optimal spacing of the fluorescent moieties in the oligonucleotide and the polymerase chosen for a particular fluorescently labeled nucleotide.
- Primer extension refers to the template directed, polymerase driven process of extending a primer oligonucleotide which is base paired to a template with nucleotide triphosphates, such that the final product is a (fully or partially) duplex DNA strand.
- a "target sequence” is that oligonucleotide sequence which is to be labeled (either covalently or non-covalently) , coupled or ligated to an optimally fluorescent moiety.
- nick-translation is catalyzed by DNA polymerase, and it is characterized by the simultaneous polymerization of new DNA and the degradation of DNA ahead of the growing site.
- a “DNA matrix (or matrices)” refers to successive layers of polynucleotides of specific structure, including a double-stranded waist and single stranded, free arms at the molecule ends, formed by hybridization of the arms to adjacent molecule arms. Such matrices are described in U.S. Patent Nos. 5,175,270 and 5,487,973, which are incorporated herein by reference. "Specific activity” refers to that amount of radiolabel present per unit mass of labeled compound, and it is usually expressed in units of Curies (Ci) per millimole (mmol) .
- the process known as a "Southern blot" enables the detection of specific sequences of a nucleic acid to be detected by a labeled probe.
- the label is radioactive the result is visualized by autoradiography.
- the restricted DNA fragments are denatured in a gel and blotted onto a sheet of membrane nitrocellulose or nylon by capillary action or electrophoretic transfer in a manner that preserves the original pattern.
- the sheet is incubated in a solution containing labeled probe (i.e., complementary DNA or RNA). Once the homologous sequences have had time to anneal, the membrane is washed free of unhybridized probe.
- RNA fragments bear ho ology to the nucleotide sequence on the probe.
- a "Northern blot" is the analogous process whereby specific sequences of RNA are detected by a labeled probe.
- the RNA is blotted onto a membrane, and the sheet is incubated in a solution containing labeled probe. After the complementary sequences have annealed, the medium is washed free of unhybridized probe and the label is detected. The result will indicate which RNA fragments bear homology to the nucleotide sequence on the probe.
- a nucleic acid "dot blot" is produced when a nucleic acid in solution is detected by spotting the solution on a membrane and detected as in a Southern or Northern blot. Dot blots can be used to quantitate the amount of nucleic acid in an extract.
- Random priming refers to the process whereby double stranded DNA is denatured in the presence of random primers, and unlabeled nucleotide triphosphates, 32 P-labeled nucleotide triphosphates and polymerase are added to initiate elongation of the primer, followed by denaturation to release labeled probe.
- a "microtitre plate assay” refers to the detection of an antigen-antibody, dye-substrate or probe- target interaction between a solution of unknown concentration of antigen, protein or DNA/RNA.
- the unknown solution is placed in a microtitre plate, which consists of individual wells for small volumes (usually no more than 200 ⁇ l) , and is reacted with an antibody solution, dye or probe of known concentration.
- the degree of interaction between the reactant and unknown solution is indicative of the concentration of the solute present in the unknown solution.
- the interaction can be assessed by fluorescence, ultra-violet absorption, or reaction with a secondary antibody solution.
- the method of the present invention generates labeled oligonucleotides with a known number and spacing of fluorescent moieties in the sequence.
- the oligonucleotides of the present invention may be represented by the formula: where n is an integer from 20 to 1000; wherein all nucleotides in the sequence are capable of forming a base pair with an optimally fluorescent dye-conjugated nucleotide triphosphate.
- the corresponding optimally fluorescent oligonucleotide may be represented by the formula:
- N f (N f ) n N f where n is an integer from 20 to 1000; wherein N f represents an optimally fluorescent nucleotide in the sequence.
- oligonucleotides of the present invention may be represented by the formula:
- nucleotide N fc is capable of forming a base pair with an optimally fluorescent dye- conjugated nucleotide triphosphate, and nucleotide, N, is not capable of forming such base pairs.
- the corresponding optimally fluorescent oligonucleotide may be represented by the formula: where n is an integer from 20 to 1000, and m is an integer from 1 to 11; wherein nucleotide N f represents a fluorescently labeled nucleotide in the sequence, and nucleotide N, is not labeled fluorescently.
- the labeling of the target sequence with the fluorescent moiety can be done prior to or during the incorporation of the target sequence to the oligonucleotide.
- the target sequence can be attached to the fluorescently labeled oligonucleotide by primer extension or ligation.
- the fluorescent moieties can be incorporated with the target sequence during the polymerization reaction between the target and an appropriate template, with the addition of dye-conjugated nucleotide triphosphates (NTPs) in addition to unlabeled NTPs, by cloning or randomer extension.
- NTPs dye-conjugated nucleotide triphosphates
- the process begins with the determination of the optimal spacing and preferred polymerase for each dye-NTP conjugate.
- a primer sequence preferably 6-40 bases long
- multiple template sequences will be required.
- the template sequences (20- 100 bases) will have a primer binding region and downstream from the primer binding region the appropriate nucleotide ("G” for "C” conjugated dyes, "A” for "U” conjugated dyes, etc.) spaced every base (polyhomonucleotide in a first template sequence) , every other base in a second template sequence, every third base, every forth base, every fifth base, every sixth base, every seventh base, every eight base, every ninth base, every tenth base or every eleventh base.
- nucleotide repeat region The repetition of nucleotide in this manner is referred to herein as a nucleotide repeat region, and it can be represented by the following formula: , ⁇ where N represents a nucleotide which is not capable of forming a base pair to a dye-conjugated nucleotide; N fc represents the nucleotide which is capable of forming a base pair to a dye-conjugated nucleotide, or that which is directly conjugated to the fluorescent dye.
- the spacing of the dye-conjugated nucleotides within the nucleotide repeat region should be as close as possible without quenching the fluorescence signal of the individual moieties.
- the intervening sequence can be repeated sequence, semi-repeated, or random sequence selected from the three non-basepairing (to the dye-NTP) bases.
- the primary constraint on the intervening sequence is the absence of self homology, either intertemplate or intratemplate to minimize non-specific priming events.
- a single set of primers is sufficient for determining the optimal spacing for any dye-NTP conjugate.
- the primer should be radiolabeled, preferably with 32 P, to high specific activity, and the actual specific activity should be determined by counting an aliquot of the radiolabeled primer and measuring the optical density at 260nm. The actual determination of the specific activity may be omitted if the optimal spacing is the only information desired from the experiment; however, determination of the specific activity allows for rapid subsequent determination of the specific fluorescence.
- the 5'- 32 P labeled primer and templates (in a separate reaction for each template sequence) should be mixed in approximately stoichiometric ratios, and allowed to anneal.
- the annealing process can be done in any buffer conducive to the formation of nucleic acid hybrids, such as lOOmM Tris-HCl, pH 8.0,
- annealed primer-template 200mM NaCl, ImM EDTA. After annealing, the sample can be precipitated with ethanol and resuspended in water, or alternatively used directly in the polymerization assay. An aliquot, approximately l ⁇ g, of annealed primer-template should then be added to a series of reactions using multiple polymerases, such as SEQUENASETM from Amersham Life Sciences, Klenow fragment of DNA Poll, Taq Polymerase, Pyrostase, and other commercially available polymerases. The reaction should take place in the optimized buffer for each particular polymerase (as determined by the manufacturer) .
- SEQUENASETM SEQUENASETM from Amersham Life Sciences
- Klenow fragment of DNA Poll Klenow fragment of DNA Poll
- Taq Polymerase Klenow fragment of DNA Poll
- Pyrostase Pyrostase
- the reaction should also contain the dye-NTP, and unlabeled NTPs at a concentration of 20 ⁇ M to 2mM (excluding the NTP which is already added as a part of the dye-NTP conjugate) .
- Each polymerase is capable of recognizing and incorporating the dye-NTP conjugates into the polymerization reaction to a different degree, and the choice of enzyme may significantly affect the specific fluorescence of the labeled probe.
- the labeled oligonucleotides should be purified away from the unincorporated nucleotide triphosphates. The purification can be accomplished by ethanol precipitation, size exclusion chromatography, gel electrophoresis or another method.
- the purified labeled oligonucleotides should be quantitated by scintillation counting or, if sufficiently large quantities are available, by measuring the optical density at 260nm and at the wavelength of maximum absorbance of the dye moiety.
- the specific fluorescence of the purified labeled oligonucleotides is then determined.
- a known aliquot of the labeled oligonucleotide is diluted in reagent grade water and the amount of fluorescence determined with a fluorometer, preferably a variable slit spectrofluoro eter.
- the reaction mixture showing the greatest specific fluorescence is selected as the optimal labeling method for that particular dye-NTP conjugate.
- the methods of the present invention can be used for labeling ribonucleotide sequences, in which case, RNA polymerase and labeled ribonucleotides would be used in the synthesis of optimally labeled oligonucleotides. Labeling of a Target Sequence
- target sequences may be labeled by the optimally labeled oligonucleotide by ligation of the target sequence to fluorescently labeled nucleotides, cloning the target sequence adjacent to the optimal spacing sequence or by "randomer" extension reaction.
- Labeling by ligation is accomplished by first synthesizing and purifying an optimally labeled nucleic acid (20 bases to 2 kilobases) .
- the target sequence for labeling with the fluorescently labeled oligonucleotide is nicked into small pieces, which average 30-70 bases, by chemical degradation or by treatment with nuclease such as DNAse I or a restriction enzyme.
- nuclease such as DNAse I or a restriction enzyme.
- Approximately equal weights of fluorescently labeled oligonucleotide typically 50ng to 5 ⁇ g in 50-100 ⁇ l total reaction volume
- target sequence are reacted in ligation buffer as recommended by the ligase enzyme manufacturer.
- the relative success of the ligation step can be assessed by gel electrophoresis.
- the ligated material can be directly used in hybridization assays or, if desired, purified by precipitation, size fractionation, gel electrophoresis, antigen-specific binding, or another method. Labeling
- the basis of this labeling technique is the use of a short (6-12 base) random sequence at the 3' end of the optimally labeled oligonucleotide.
- the initial labeling reaction of the template with fluorescent compound is modified such that the template molecule is designed to have a 5' overhang (the extension region for incorporation of dye-NTP) as well as a 3' overhang of 6- 200 bases with the most 3' sequence being a random sequence of typically 6-12 bases.
- the purified labeled oligonucleotide may be used directly in the primer extension reaction or preferably crosslinked with trimethylpsoralen prior to use in the target labeling reaction.
- the target labeling reaction consists of denaturing the desired target sequence, adding the polymerase, an excess of labeled-primer molecules and the appropriate NTPs for the desired polymerase (i.e. dATP, TTP, dCTP, dGTP for use with Klenow polymerase) , in the appropriate buffer.
- NTPs for the desired polymerase i.e. dATP, TTP, dCTP, dGTP for use with Klenow polymerase
- Some of the 3' ends of the fluorescently labeled randomer will serve as primers on the target molecule thereby being extended during the polymerization process and generating molecules having a 3' end complementary to the target molecule and an optimally labeled 5' end.
- subsequent polymerase labeling cogenerates polynucleotides having the optimally labeled sequence and the target sequence.
- the polymerase may be an RNA polymerase, such as T7 RNA polymerase for use with ribonucleotide triphosphates.
- the polymerase may also be a DNA polymerase and the labeling performed by specific primer extension or via random priming methods.
- the labeled nucleic acids may be used as probes for a particular sequence wherever highly fluorescent nucleic acid probes are desired, e.g., in known nucleic acid assay methods such as dot blot, Southern blot or Northern blot, etc.
- the fluorescently labeled oligonucleotides may be used for in situ hybridization techniques, wherein the sequence of interest is present in only a small number of cells within a large mixed population. Such sequences may be undetectable in tissue extracts due to the presence of interfering sequences from surrounding tissue.
- In situ hybridization may be used to: (1) identify sites of gene expression; (2) analyze the tissue distribution of transcription; (3) identify and localize viral infection; (4) follow changes in specific mRNA synthesis; and (5) aid in chromosome mapping.
- the present invention can provide increased specific fluorescence and therefore, enhanced sensitivity when compared to conventional methods for in situ hybridization.
- Another use for the present invention is for the enhanced detection of nucleic acid sequences in combination with DNA matrices, which are disclosed in U.S. Patent Nos. 5,175,270 and 5,487,973, and which are incorporated herein by reference.
- the DNA matrices disclosed in U.S. Patent Nos. 5,175,270 and 5,487,973 comprise successive layers of polynucleotides having both single and double-stranded regions.
- oligonucleotide probes of the present invention can be hybridized to the non-annealed, free, single-stranded arms of the DNA matrices, and the resulting fluorescently labeled DNA matrices can be useful in the assay of a wide variety of nucleic acid sequences including those associated with pathogenic bacteria and viruses.
- the present invention can be used in a microtitre plate assay system based on fluorescence, wherein the high specific fluorescence provided by the optimally fluorescent oligonucleotide probes would enhance and facilitate the detection of the fluorescent moiety in the assay.
- the polymerase and optimal spacing was determined for dCTP-Cy3 incorporation.
- the fluorescently labeled strand be longer than the template strand so the primer sequence had a 5' overhang relative to the template strand.
- the template therefore utilized only three bases, "G", “A”, and “T”, so that "back” reaction (extension of the template sequence on the primer sequence) could be blocked by omitting dGTP from the reaction buffer.
- the reaction By designing the reaction to allow a 5' overhang on the primer strand, subsequent strand separation could be readily achieved by denaturing gel electrophoresis, since in the post reaction, the extension product is longer than the template sequence.
- the templates were 41mers designated a(+)-2C, a(+)-3C and a(+)-4C, each designed to incorporate the dCTP-Cy3 dye every other, every third, or every fourth base respectively.
- the primer sequence was a 31mer designed to hybridize with the template strand over 14 bases. Full extension of the primer was expected to yield a 58mer, with 27 bases added by the polymerase reaction.
- the primer sequence was 5' labeled with 32 P by ⁇ 32 P-ATP (ICN).
- Radiochemicals Cat # 35020 100 uCi/ reaction and 10U of polynucleotide kinase (Boehringer Mannheim Biochemicals) in the manufacturer's supplied reaction buffer and recommended reaction time.
- the primer was purified essentially free of unincorporated nucleotide by size exclusion chromatography (select-D-G25 Column 5'- 3'®, Boulder, CO) as recommended by the manufacturer, and it had a specific activity of 31,180 cpm/ng.
- the primer was stored in aliquots, each at a concentration of 62.2 ng/ ⁇ l (as determined by the OD 260 of 58.3 ⁇ l in 1 ml of reagent grade water) in lOOmM Tris-HCl, pH 8.0, containing 200mM NaCl and ImM EDTA.
- the annealing reaction was carried out by reacting 25 ⁇ l template oligonucleotide (5 ⁇ g, 0 cpm) and 49 ⁇ l 32 P labeled primer (3.0 ⁇ g, 39,540,000 cpm) in 24 ⁇ l reagent grade water containing 2.0 ⁇ l 5M NaCl (final concentration of lOOmM NaCl) .
- the reaction was cooled from 95°C to room temperature over 15 minutes in a 1L beaker.
- the subsequent polymerase extension reaction was carried out by combining lO.O ⁇ l of the aforementioned annealed oligonucleotide reaction mixture, lO ⁇ l 5X reaction buffer (supplied by the manufacturer of the polymerase) , l ⁇ l dATP, l ⁇ l dTTP (each lOmM, supplied by Boehringer Mannheim), 5.0 ⁇ l dCTP-Cy3 (ImM, supplied by Biological Detection Systems) , 22 ⁇ l reagent grade water and l ⁇ l SEQUENASETM (USB United States Biologicals, 10 units) or Klenow fragment of DNA Poll (supplied by Boehringer Mannheim, 10 units) . The reaction was complete after 1 hour at room temperature.
- each reaction was loaded on a 9% denaturing polyacryla ide gel. Following electrophoresis, the gel was dried on 3MM paper and exposed to x-ray film for autoradiography. Then, a separate aliquot from each of the reactions was loaded on a preparative 9% denaturing acrylamide gel, electrophoresed, and stained with ethidium bromide.
- the labeled (5' 32 P and 3' Cy3-CTP at varying spacing) 58mers were excised from the gel, triturated with 200 ⁇ l lOmM Tris-HCl, pH 8.0, containing ImM EDTA, and the samples were shaken overnight in 1.5ml microcentrifuge tubes at 37°C.
- the 3C optimally fluorescent oligonucleotide can be used to label the outer layer of polynucleotides of a DNA matrix, through their non-annealed, free, single-stranded arms.
- the fluorescently labeled DNA matrix can be used to recognize the multiple DNA arms of the sequence bound to a smaller bead, and to supply an easily measured mass to the assay system.
- the DNA bead matrix is assembled as described in U.S. Patent No. 5,487,973. Sequential additions of matrix monomers leads to a DNA matrix with k layers (k-Mmer) .
- the double-stranded, unpurified 3C optimally fluorescent oligonucleotide (which has a 5' single-stranded overhang) is added as the final addition to the k-Mraer, yielding a DNA bead matrix having optimally fluorescent single-stranded arms.
Abstract
Description
Claims
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA002229017A CA2229017A1 (en) | 1996-06-04 | 1997-06-02 | Optimally fluorescent oligonucleotides |
EP97927859A EP0857221A4 (en) | 1996-06-04 | 1997-06-02 | Optimally fluorescent oligonucleotides |
JP10500708A JPH11510709A (en) | 1996-06-04 | 1997-06-02 | Optimal fluorescent oligonucleotide |
AU32217/97A AU718610B2 (en) | 1996-06-04 | 1997-06-02 | Optimally fluorescent oligonucleotides |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US08/657,961 | 1996-06-04 | ||
US08/657,961 US6072043A (en) | 1996-06-04 | 1996-06-04 | Optimally fluorescent oligonucleotides |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1997046703A1 true WO1997046703A1 (en) | 1997-12-11 |
Family
ID=24639340
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1997/009270 WO1997046703A1 (en) | 1996-06-04 | 1997-06-02 | Optimally fluorescent oligonucleotides |
Country Status (6)
Country | Link |
---|---|
US (3) | US6072043A (en) |
EP (1) | EP0857221A4 (en) |
JP (1) | JPH11510709A (en) |
AU (1) | AU718610B2 (en) |
CA (1) | CA2229017A1 (en) |
WO (1) | WO1997046703A1 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6203989B1 (en) | 1998-09-30 | 2001-03-20 | Affymetrix, Inc. | Methods and compositions for amplifying detectable signals in specific binding assays |
Families Citing this family (38)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6072043A (en) * | 1996-06-04 | 2000-06-06 | Polyprobe, Inc. | Optimally fluorescent oligonucleotides |
JP2003500070A (en) * | 1999-05-21 | 2003-01-07 | インビトロゲン・コーポレーション | Compositions and methods for labeling nucleic acid molecules |
CA2403911A1 (en) * | 2000-03-29 | 2001-10-04 | Polyprobe, Inc. | Quality control reagents for nucleic acid microarrays |
US7011943B2 (en) * | 2000-09-06 | 2006-03-14 | Transnetyx, Inc. | Method for detecting a designated genetic sequence in murine genomic DNA |
WO2003012147A1 (en) * | 2001-02-20 | 2003-02-13 | Datascope Investment Corp. | Method for reusing standard blots and microarrays utilizing dna dendrimer technology |
GB0112238D0 (en) * | 2001-05-18 | 2001-07-11 | Medical Biosystems Ltd | Sequencing method |
WO2003062783A2 (en) * | 2001-07-20 | 2003-07-31 | North Carolina State University | Light addressable electrochemical detection of duplex structures |
EP1438435B1 (en) * | 2001-08-31 | 2007-10-31 | Datascope Investment Corp. | Methods for blocking nonspecific hybridisations of nucleic acid sequences |
US7045319B2 (en) * | 2001-10-30 | 2006-05-16 | Ribomed Biotechnologies, Inc. | Molecular detection systems utilizing reiterative oligonucleotide synthesis |
US20030180769A1 (en) * | 2002-02-05 | 2003-09-25 | Metzker Michael L. | Substituted 4,4-difluoro-4-bora-3A,4A-diaza-s-indacene compounds for 8-color DNA sequencing |
DE10206616A1 (en) * | 2002-02-15 | 2003-09-04 | Qiagen Gmbh | Process for reducing background contamination after labeling reactions |
AU2003267960A1 (en) * | 2002-05-22 | 2004-01-06 | Marshall University | Methods, probes, and accessory molecules for detecting single nucleotide polymorphisms |
WO2003106637A2 (en) * | 2002-06-12 | 2003-12-24 | Datascope Investment Corp. | Polymeric label molecules |
DE60327775D1 (en) * | 2002-06-24 | 2009-07-09 | Exiqon As | METHODS AND SYSTEMS FOR THE DETECTION AND ISOLATION OF NUCLEIC ACID SEQUENCES |
US7476519B2 (en) * | 2002-07-19 | 2009-01-13 | Joseph Monforte | Strategies for gene expression analysis |
US20040180369A1 (en) * | 2003-01-16 | 2004-09-16 | North Carolina State University | Photothermal detection of nucleic acid hybridization |
US7514551B2 (en) * | 2003-04-03 | 2009-04-07 | Enzo Life Sciences, Inc. | Multisignal labeling reagents, and processes and uses therefor |
US9156986B2 (en) | 2003-04-03 | 2015-10-13 | Enzo Life Sciences, Inc. | Multisignal labeling reagents and processes and uses therefor |
US9696298B2 (en) | 2003-04-03 | 2017-07-04 | Enzo Life Sciences, Inc. | Multisignal reagents for labeling analytes |
KR20070012779A (en) * | 2003-10-29 | 2007-01-29 | 리보메드 바이오테그놀로지스 인코포레이티드 | Compositions, methods and detection technologies for reiterative oligonucleotide synthesis |
US20100000881A1 (en) * | 2003-10-30 | 2010-01-07 | North Carolina State University | Electrochemical detection of nucleic acid hybridization |
WO2005042783A1 (en) * | 2003-10-30 | 2005-05-12 | North Carolina State University | Temperature-jump enhanced electrochemical detection of nucleic acid hybridization |
US20060094025A1 (en) * | 2004-11-02 | 2006-05-04 | Getts Robert C | Methods for detection of microrna molecules |
US7550264B2 (en) | 2005-06-10 | 2009-06-23 | Datascope Investment Corporation | Methods and kits for sense RNA synthesis |
US20060281100A1 (en) * | 2005-06-14 | 2006-12-14 | Shen Gene G | Thiotriphosphate nucleotide dye terminators |
US20070048741A1 (en) * | 2005-08-24 | 2007-03-01 | Getts Robert C | Methods and kits for sense RNA synthesis |
US20070105124A1 (en) * | 2005-11-08 | 2007-05-10 | Getts Robert C | Methods and kits for nucleic acid amplification |
US8685899B2 (en) | 2007-02-14 | 2014-04-01 | Genisphere Inc. | Methods, reagents and kits for detection of nucleic acid molecules |
US20100190167A1 (en) * | 2007-02-14 | 2010-07-29 | Genisphere Inc. | Methods, Reagents and Kits for Detection of Nucleic Acid Molecules |
US9222936B2 (en) | 2007-04-18 | 2015-12-29 | Solulink, Inc. | Methods and/or use of oligonucleotide conjugates for suppressing background due to cross-hybridization |
WO2008137661A1 (en) * | 2007-05-03 | 2008-11-13 | Helicos Biosciences Corporation | Methods and compositions for sequencing a nucleic acid |
EP2225561A4 (en) | 2007-11-28 | 2011-09-14 | Great Basin Scient | Methods and compositions for signal enhancement using multivalent interactions |
US20110206611A1 (en) | 2010-02-24 | 2011-08-25 | Genisphere, Llc | DNA Dendrimers as Thermal Ablation Devices |
US9651549B2 (en) | 2012-07-13 | 2017-05-16 | Genisphere, Llc | Lateral flow assays using DNA dendrimers |
US20180187246A1 (en) * | 2015-07-15 | 2018-07-05 | Universiteit Gent | Probes and a methylation in situ hybridization assay |
WO2019200326A1 (en) | 2018-04-13 | 2019-10-17 | Rarecyte, Inc. | Kits for labeling of biomarkers and methods of using the same |
CA3130693A1 (en) | 2019-02-19 | 2020-08-27 | Ultima Genomics, Inc. | Linkers and methods for optical detection and sequencing |
US11807851B1 (en) | 2020-02-18 | 2023-11-07 | Ultima Genomics, Inc. | Modified polynucleotides and uses thereof |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5403708A (en) * | 1992-07-06 | 1995-04-04 | Brennan; Thomas M. | Methods and compositions for determining the sequence of nucleic acids |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5175270A (en) * | 1986-09-10 | 1992-12-29 | Polyprobe, Inc. | Reagents for detecting and assaying nucleic acid sequences |
US5652099A (en) * | 1992-02-12 | 1997-07-29 | Conrad; Michael J. | Probes comprising fluorescent nucleosides and uses thereof |
WO1995025179A1 (en) * | 1994-03-17 | 1995-09-21 | University Of Massachusetts Medical Center | Detection of trinucleotide repeats by in situ hybridization |
US6072043A (en) * | 1996-06-04 | 2000-06-06 | Polyprobe, Inc. | Optimally fluorescent oligonucleotides |
-
1996
- 1996-06-04 US US08/657,961 patent/US6072043A/en not_active Expired - Fee Related
-
1997
- 1997-06-02 EP EP97927859A patent/EP0857221A4/en not_active Withdrawn
- 1997-06-02 AU AU32217/97A patent/AU718610B2/en not_active Ceased
- 1997-06-02 JP JP10500708A patent/JPH11510709A/en not_active Ceased
- 1997-06-02 CA CA002229017A patent/CA2229017A1/en not_active Abandoned
- 1997-06-02 WO PCT/US1997/009270 patent/WO1997046703A1/en not_active Application Discontinuation
- 1997-08-12 US US08/909,539 patent/US6046038A/en not_active Expired - Lifetime
-
2001
- 2001-07-23 US US09/911,039 patent/US6762292B2/en not_active Expired - Lifetime
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5403708A (en) * | 1992-07-06 | 1995-04-04 | Brennan; Thomas M. | Methods and compositions for determining the sequence of nucleic acids |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6203989B1 (en) | 1998-09-30 | 2001-03-20 | Affymetrix, Inc. | Methods and compositions for amplifying detectable signals in specific binding assays |
US6806047B2 (en) | 1998-09-30 | 2004-10-19 | Affymetrix, Inc. | Methods and compositions for amplifying detectable signals in specific binding assays |
Also Published As
Publication number | Publication date |
---|---|
US6046038A (en) | 2000-04-04 |
EP0857221A1 (en) | 1998-08-12 |
AU718610B2 (en) | 2000-04-20 |
US20020012972A1 (en) | 2002-01-31 |
US6762292B2 (en) | 2004-07-13 |
CA2229017A1 (en) | 1997-12-11 |
EP0857221A4 (en) | 2004-05-12 |
AU3221797A (en) | 1998-01-05 |
US6072043A (en) | 2000-06-06 |
JPH11510709A (en) | 1999-09-21 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US6072043A (en) | Optimally fluorescent oligonucleotides | |
US6054274A (en) | Method of amplifying the signal of target nucleic acid sequence analyte | |
CA2026280C (en) | Biotin-labelled dna by polymerase chain reaction and detection thereof | |
EP0359789B1 (en) | Amplification and detection of nucleic acid sequences | |
JP2788034B2 (en) | Amplification method for polynucleotide assay | |
CA2662837C (en) | Methods and substances for isolation and detection of small polynucleotides | |
CA2537810C (en) | Nucleic acid detection assay | |
US5858731A (en) | Oligonucleotide libraries useful for producing primers | |
US20020127575A1 (en) | Partially double-stranded nucleic acids, methods of making, and use thereof | |
US20040137484A1 (en) | Nucleic acid amplification methods | |
KR20010111020A (en) | Isometric primer extension method and kit for detection and quantification of specific nucleic acid | |
US5663062A (en) | Oligonucleotide libraries useful for producing primers | |
US20060223075A1 (en) | Unique sequence hybridization probes (USP) | |
EP1017855B1 (en) | Methods of synthesizing polynucleotides by ligation of multiple oligomers | |
US20050095606A1 (en) | Partially double-stranded nucleic acids, methods of making, and use thereof | |
WO2005060618A2 (en) | Enzyme-free isothermal exponential amplification of nucleic acids and nucleic acid analog signals | |
US20020031776A1 (en) | Enzymatic labeling and detection of DNA hybridization probes | |
WO1994002648A1 (en) | Gap-filling nucleic acid amplification and detection | |
AU2004270756B2 (en) | Nucleic acid detection assay | |
AU723602B2 (en) | Biotin-labelled DNA by polymerase chain reaction and detection thereof | |
JP3145169B2 (en) | Nucleic acid detection method and kit | |
US20050202461A1 (en) | Method for converting generic nucleic acid priming sequences | |
EP0620861A1 (en) | Restriction amplification assay | |
Marras | 2 Artificial Hybridization Probes | |
AU2001297868A1 (en) | Partially double-stranded nucleic acids, methods of making, and use thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AM AT AU BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE GH HU IL IS JP KE KG KP KR KZ LK LR LT LU LV MD MG MN MW MX NO NZ PL PT RO RU SD SE SG SI SK TJ TM TT UA UZ VN YU |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): GH KE LS MW SD SZ UG AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG |
|
ENP | Entry into the national phase |
Ref document number: 2229017 Country of ref document: CA Ref country code: CA Ref document number: 2229017 Kind code of ref document: A Format of ref document f/p: F |
|
WWE | Wipo information: entry into national phase |
Ref document number: 1997927859 Country of ref document: EP |
|
ENP | Entry into the national phase |
Ref country code: JP Ref document number: 1998 500708 Kind code of ref document: A Format of ref document f/p: F |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
WWP | Wipo information: published in national office |
Ref document number: 1997927859 Country of ref document: EP |
|
REG | Reference to national code |
Ref country code: DE Ref legal event code: 8642 |
|
WWW | Wipo information: withdrawn in national office |
Ref document number: 1997927859 Country of ref document: EP |