US20110151449A1 - Short cycle methods for sequencing polynucleotides - Google Patents
Short cycle methods for sequencing polynucleotides Download PDFInfo
- Publication number
- US20110151449A1 US20110151449A1 US13/008,468 US201113008468A US2011151449A1 US 20110151449 A1 US20110151449 A1 US 20110151449A1 US 201113008468 A US201113008468 A US 201113008468A US 2011151449 A1 US2011151449 A1 US 2011151449A1
- Authority
- US
- United States
- Prior art keywords
- nucleotide
- nucleotides
- primer
- cycle
- incorporation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Images
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
- C12Q1/6869—Methods for sequencing
- C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation
-
- 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
- C12Q1/6869—Methods for sequencing
Definitions
- the invention relates to methods for sequencing a polynucleotide, and more particularly, to methods for high throughput single molecule sequencing of target polynucleotides.
- Cancer is a disease that is rooted in heterogeneous genomic instability. Most cancers develop from a series of genomic changes, some subtle and some significant, that occur in a small subpopulation of cells. Knowledge of the sequence variations that lead to cancer will lead to an understanding of the etiology of the disease, as well as ways to treat and prevent it.
- An essential first step in understanding genomic complexity is the ability to perform high-resolution sequencing.
- nucleic acid sequencing Various approaches to nucleic acid sequencing exist.
- One conventional way to do bulk sequencing is by chain termination and gel separation, essentially as described by Sanger et al., Proc Natl Acad Sci USA, 74(12): 5463-67 (1977). That method relies on the generation of a mixed population of nucleic acid fragments representing terminations at each base in a sequence. The fragments are then run on an electrophoretic gel and the sequence is revealed by the order of fragments in the gel.
- Another conventional bulk sequencing method relies on chemical degradation of nucleic acid fragments. See, Maxam et al., Proc. Natl. Acad. Sci., 74: 560-564 (1977).
- methods have been developed based upon sequencing by hybridization.
- optical signaling When optical signaling is used as the detection means, conventional optics are able to reliably distinguish one from two identical bases, and sometimes two from three, but rarely more than three. Thus, single molecule sequencing using fluorescent labels in a homopolymer region typically results in a signal that does not allow accurate determination of the number of bases in the region.
- nucleotide analogues that have a modification at the 3′ carbon of the sugar that reversibly blocks the hydroxyl group at that position.
- the added nucleotide is detected by virtue of a label that has been incorporated into the 3′ blocking group.
- the blocking group is cleaved, typically, by photochemical means to expose a free hydroxyl group that is available for base addition during the next cycle.
- the invention provides methods for high throughput single molecule sequencing.
- the invention provides methods for controlling at least one parameter of a nucleotide extension reaction in order to regulate the rate at which nucleotides are added to a primer.
- the invention provides several ways of controlling nucleic acid sequence-by-synthesis reactions in order to increase the resolution and reliability of single molecule sequencing. Methods of the invention solve the problems that imaging systems have in accurately resolving a sequence at the single-molecule level. In particular, methods of the invention solve the problem of determining the number of nucleotides in a homopolymer stretch.
- Methods of the invention generally contemplate terminating sequence-by-synthesis reactions prior to completion in order to obtain increased resolution of individual nucleotides in a sequence.
- this requires exposing nucleotides to a mixture comprising a template, a primer, and a polymerase under conditions sufficient for only limited primer extension. Reactions are conducted under conditions such that it is statistically unlikely that more than 1 or 2 nucleotides are added to a growing primer strand in any given incorporation cycle.
- An incorporation cycle comprises exposure of a template/primer to nucleotides directed at the base immediately downstream of the primer (this may be all four conventional nucleotides or analogs if the base is not known) and washing unhybridized nucleotide.
- Nucleotide addition in a sequence-by-synthesis reaction is a stochastic process. As in any chemical reaction, nucleotide addition obeys the laws of probability. Methods of the invention are concerned with controlling the rate of nucleotide addition on a per-cycle basis. That is, the invention teaches ways to control the rate of nucleotide addition within an extension cycle given the stochastic nature of the extension reaction itself. Methods of the invention are intended to control reaction rates within the variance that is inherent in a reaction that is fundamentally stochastic. Thus, the ability to control, according to the invention, base addition reactions such that, on average, no more than two bases are added in any cycle takes into account the inherent statistics of the reactions.
- the invention thus teaches polynucleotide sequence analysis using short cycle chemistry.
- One embodiment of the invention provides methods for slowing or reversibly inhibiting the activity of polymerase during a sequencing-by-synthesis reaction.
- Other methods teach altering the time of exposure of nucleotides to the template-primer complex.
- Still other methods teach the use of physical blockers that temporarily halt or slow polymerase activity and/or nucleotide addition.
- any component of the reaction that permits regulation of the number of labeled nucleotides added to the primer per cycle, or the rate at which the nucleotides are incorporated and detected per cycle is useful in methods of the invention.
- Additional components include, but are not limited to, the presence or absence of a label on a nucleotide, the type of label and manner of attaching the label; the linker identity and length used to attach the label; the type of nucleotide (including, for example, whether such nucleotide is a dCTP, dTTP, dGTP or dUTP; a natural or non-natural nucleotide, a nucleotide analogue, or a modified nucleotide); the “half-life” of the extension cycle (where one half-life is the time taken for at least one incorporation to occur in 50% of the complementary strands); the local sequence immediately 3′ to the addition position; whether such base is the first, second, third, etc.
- the base added added; the type of polymerase used; the particular batch characteristics of the polymerase; the processivity of the polymerase; the incorporation rate of the polymerase; the number of wash cycles (i.e., the number of times a nucleotide is introduced to the reaction then washed out); the number of target nucleic acids in the reaction; the temperature of the reaction and the reagents used in the reaction.
- a nucleic acid template is exposed to a primer capable of hybridizing to the template and a polymerase capable of catalyzing nucleotide addition to the primer.
- a labeled nucleotide is introduced for a period of time that is statistically insufficient for incorporation of more than about 2 nucleotides per cycle.
- Nucleotide exposure may also be coordinated with polymerization inhibition such that, on average, 0, 1, or 2 labeled nucleotides are added to the primer, but that 3 labeled nucleotides are almost never added to the primer in each cycle.
- the exposure time, during which labeled nucleotides are exposed to the template-primer complex is statistically insufficient for incorporation of more nucleotides than are resolvable by a detection system used to detect incorporation.
- the invention also contemplates performing a plurality of base incorporation cycles.
- Each cycle comprises exposing a template nucleic acid to a labeled nucleotide that is not a chain-terminating nucleotide.
- the labeled nucleotide is incorporated into a primer hybridized to the template nucleic acid if the nucleotide is capable of hybridizing to the template nucleotide immediately upstream of the primer and there is about a 99% probability that two or fewer of said nucleotides are incorporated into the same primer strand per cycle.
- Incorporated nucleotides are then identified.
- Methods of the invention also make use of differential base incorporation rates in order to control overall reaction rates. For example, the rate of incorporation is lower for a second nucleotide given incorporation of a prior nucleotide immediately upstream of the second. This effect is magnified if the first nucleotide comprises a label or other group that hinders processivity of the polymerase.
- the rate of incorporation is lower for a second nucleotide given incorporation of a prior nucleotide immediately upstream of the second. This effect is magnified if the first nucleotide comprises a label or other group that hinders processivity of the polymerase.
- the invention may also be conducted using a plurality of primer extension cycles, wherein each cycle comprises exposing a target nucleic acid to a primer capable of hybridizing to the target, thereby forming a primed target; exposing the primed target to a labeled nucleic acid in the presence of a nucleic acid polymerase, coordinating transient inhibition of the polymerase and time of exposure to the labeled nucleotide such that it is statistically likely that at least one of said labeled nucleic acid is incorporated in the primer, but statistically unlikely that more than two of the labeled nucleotide are incorporated in the primer.
- methods of the invention comprise conducting a cycle of template-dependent nucleic acid primer extension in the presence of a polymerase and a labeled nucleotide; inhibiting polymerase activity such that it is statistically unlikely that more than about 2 nucleotides are incorporated into the same primer strand in the cycle; washing unincorporated labeled nucleotide away from the template; detecting any incorporation of the labeled nucleotide; neutralizing label in any incorporated labeled nucleotide; removing the inhibition; repeating the foregoing steps; and compiling a sequence based upon the sequence of nucleotides incorporated into the primer.
- the invention provides a method comprising exposing a nucleic acid template to a primer capable of hybridizing to a portion of the template in order to form a template/primer complex reaction mixture; adding a labeled nucleotide in the presence of a polymerase to the mixture under conditions that promote incorporation of the nucleotide into the primer if the nucleotide is complementary to a nucleotide in the template that is downstream of said primer; coordinating removal of the labeled nucleotide and inhibition of the polymerase so that no more than about 2 nucleotides are incorporated into the same primer; identifying labeled nucleotide that has been incorporated into said primer; repeating the foregoing steps at least once; and determining a sequence of the template based upon the order of the nucleotides incorporated into the primer.
- the method comprises exposing a template nucleic acid to a primer capable of hybridizing to a portion of the template upstream of a region of the template to be sequenced; introducing a labeled nucleic acid and a polymerase to the template under conditions wherein the labeled nucleic acid will be incorporated in the primer if the labeled nucleic acid is capable of hybridizing with base downstream of the primer; and controlling the rate of the incorporation by limiting the time of exposure of the labeled nucleic acid to the template or by inhibiting the polymerase at a predefined time after exposure of the template to the labeled nucleotide; detecting incorporation of the labeled nucleotide into the primer; and identifying the nucleotide in the template as the complement of labeled nucleotide incorporated into the primer.
- methods of the invention comprise exposing a target polynucleotide to a primer capable of hybridizing to the polynucleotide, extending the primer in the presence of a polymerizing agent and one or more extendible nucleotides, each comprising a detectable label.
- the polymerizing agent is exposed to a cofactor (i.e., any agent that decreases or halts polymerase activity), and the incorporation of label is detected.
- the steps of extending the primer and exposing the polymerizing agent to a cofactor may be performed simultaneously, or may be performed in separate steps.
- the method further comprises inactivating the cofactor, thereby reversing its effect on the polymerizing agent. Modes of inactivation depend on the cofactor. For example, where the cofactor is attached to the nucleotide, inactivation can typically be achieved by cleaving the cofactor from the nucleotide.
- Methods of the invention also address the problem of reduced detection due to a failure of some strands in a given cycle to incorporate labeled nucleotide.
- a certain number of strands fail to incorporate a nucleotide that should be incorporated based upon its ability to hybridize to a nucleotide present in the template.
- the strands that fail to incorporate a nucleotide in a cycle will not be prepared to incorporate a nucleotide in the next cycle (unless it happens to be the same as the unincorporated nucleotide, in which case the strand will still lag behind unless both nucleotides are incorporated in the same cycle).
- the invention overcomes this problem by exposing a template/primer complex to a labeled nucleotide that is capable of hybridizing to the template nucleotide immediately downstream of the primer. After removing unbound labeled nucleotide, the sample is exposed to unlabeled nucleotide, preferably in excess, of the same species. The unlabeled nucleotide “fills in” the positions in which hybridization of the labeled nucleotide did not occur.
- the labeled nucleotide comprises a label that impedes the ability of polymerase to add a downstream nucleotide, thus temporarily halting the synthesis reaction until unlabeled nucleotide can be added, at which point polymerase inhibition is removed and t he next incorporation cycle is conducted
- One feature of this embodiment is that a sequence is compiled based upon the incorporation data, while allowing maximum strand participation in each cycle.
- methods of the invention are useful for identifying placeholders in some strands in a population of strands being sequenced. As long as there are no more than two consecutive placeholders in any one strand, the invention has a high tolerance for placeholders with little or no effect on the ultimate sequence determination.
- Methods of the invention are also useful for identifying a single nucleotide in a nucleic acid sequence.
- the method comprises the steps of sequentially exposing a template-bound primer to a labeled nucleotide and an unlabeled nucleotide of the same type in the presence of a polymerase under conditions that allow template-dependent primer extension; determining whether the first nucleotide is incorporated in the primer at a first position; repeating the sequentially exposing step using subsequent labeled and unlabeled nucleotides until a nucleotide is identified at the first position.
- FRET fluorescence resonance energy transfer
- the nucleotide is identified based upon knowledge of which nucleotide species contained the acceptor.
- the invention also provides methods for identifying a placeholder in a nucleic acid sequence using FRET.
- a nucleic acid primer is hybridized to a target nucleic acid at a primer binding site in the target.
- the primer comprises a donor fluorophore.
- the hybridized primer is exposed to a first nucleotide comprising an acceptor fluorophore that, when incorporated into the primer, prevents further polymerization of the primer. Whether there is fluorescent emission from the donor and the acceptor is determined, and a placeholder in the nucleic acid sequence is identified as the absence of emission in both the donor and the acceptor.
- the method comprises hybridizing a nucleic acid primer comprising a donor fluorophore to a target nucleic acid at a primer binding site in the target; exposing the hybridized primer to a first nucleotide comprising an acceptor fluorophore that, when incorporated into the primer, prevents further polymerization of the primer; detecting the presence or absence of fluorescent emission from each of the donor and the acceptor; identifying a nucleotide that has been incorporated into the primer via complementary base pairing with the target as the presence of fluorescent emission from the acceptor; identifying a sequence placeholder as the absence of fluorescent emission from the donor and the acceptor; and repeating the exposing, detecting, and each of the identifying steps, thereby to compile a sequence of the target nucleic acid based upon the sequence of the incorporated nucleotides and the placeholders.
- the invention is useful in sequencing any form of polynucleotides, such as double-stranded DNA, single-stranded DNA, single-stranded DNA hairpins, DNA/RNA hybrids, RNAs with a recognition site for binding of the polymerizing agent, and RNA hairpins.
- the invention is particularly useful in high throughput sequencing of single molecule polynucleotides in which a plurality of target polynucleotides are attached to a solid support in a spatial arrangement such that each polynucleotides is individually optically resolvable.
- each detected incorporated label represents a single polynucleotide.
- FIG. 1 shows asynchronous single molecule sequencing
- FIG. 2 are screenshots showing data from short cycle sequencing with long homopolymer regions.
- FIG. 2 a shows full cycle sequencing used to analyze 10 target polynucleotides in a simulated synthesis of their complementary strands using cycle periods of 10 half-lives and repeating the wash cycles 12 times.
- FIG. 2 b shows a short cycle sequencing to analyze 10 target polynucleotides by simulating the synthesis of their complementary strands using short cycle periods of 0.8 half-life periods and repeating the wash cycles 60 times.
- FIG. 3 shows a short cycle embodiment for analyzing 200 target polynucleotides in a simulated synthesis of their complementary strands using short cycle periods of 0.8 half-life periods and repeating the wash cycles 60 times.
- FIG. 4 shows a statistical analysis of incorporation, showing that polymerizing agent may incorporate repeat labeled nucleotides less readily than the first labeled nucleotide.
- FIG. 5 shows a simulation showing the effect of decreasing the activity rate of the polymerizing agent and lengthening half-lives on the cycle period.
- FIG. 6 shows the number of cycles needed with cycle periods of various half-lives taking into account stalling factors of two (squares), five (triangles) and 10 (crosses), in order to obtain over 25 incorporations in over 80% of target homopolymers, with at least a 97% chance of incorporating two or less nucleotides per cycle (or a smaller than 3% chance of incorporating more than 2 nucleotides per cycle).
- FIG. 7 is a series of screenshots showing the effects of altering reaction conditions on the incorporation of nucleotides in a single molecule sequencing by synthesis reaction.
- the invention provides methods for high throughput single molecule sequencing.
- one or more parameters of a sequencing-by-synthesis reaction are preselected such that the incorporation of, preferably, a single nucleotide on a primed target template is optically detectable.
- the preselected parameters regulate the rate at which the nucleotides are incorporated, and the rate at which the incorporated nucleotides are detected.
- the nucleotides are individually detected either as they are incorporated or shortly thereafter, essentially in “real-time.
- the preselected parameters permit the regulation of the number of nucleotides incorporated during a single extension cycle.
- the extension cycle is stopped short at a predetermined point at which, on average, only 0, 1, 2, or 3 nucleotides have been incorporated into the primer, rather than permitting the reaction to run to near or full completion in each cycle.
- Short cycle methods increase the resolution of individual nucleotides incorporated into the primer, but can decrease the yield of target templates successfully incorporating a nucleotide in a single extension cycle.
- nucleotides may be allowed to react in the presence of a polymerizing agent until at least one becomes incorporated into at least 99% of the complementary strands. This would produce a yield of (0.99) n ⁇ 100% for a complementary strand extended by n nucleotides.
- Obtaining incorporation in 99% of the complementary strands requires a period of several half-lives of the incorporation reaction, where one half-life is the time taken for at least one incorporation to occur in 50% of the complementary strands.
- the more strands that complete an incorporation during each cycle the more n-mers obtained after n cycles.
- short cycle methods rely on a period of only a limited number of half-lives of exposure to nucleotides, thus resulting in fewer target templates having incorporated a nucleotide in the short extension cycle.
- the short sequencing cycles provided by methods of the invention allow asynchronous analysis of polynucleotides.
- an incorporation reactions fails to occur on a particular target polynucleotide, it can be completed in a later cycle without producing erroneous information, or interfering with data from other target molecules being analyzed in parallel.
- a cytosine (“C”) incorporates into the extension product of one copy of a target polynucleotide, but fails to incorporate into the other copy.
- short cycle methods according the invention permit the detection of, for example, one, two or three individual nucleotides incorporated into a primed template
- the invention overcomes the difficulty posed by homopolymer regions of a template sequence. While detection techniques may be able to quantify signal intensity from a smaller number of incorporated nucleotides of the same base-type, for example two or three incorporated nucleotides, longer runs of identical bases may not permit quantification due to increasing signal intensity. That is, it may become difficult to distinguish n bases from n+1 bases, where the fractional increase in signal intensity from the (n+1)′h base is small relative to the signal intensity from the already-incorporated n bases.
- imaging systems known in the art can reliably distinguish the difference in signal intensity between one versus two fluorescent labeling moieties on consecutively-incorporated nucleotides.
- Other imaging systems can reliably distinguish the difference in signal intensity between two versus three fluorescent labeling moieties on consecutively-incorporated nucleotides.
- an extension cycle comprising a labeled nucleotide is followed by an extension cycle using an unlabeled nucleotide of the same type so that the position in each of the target template in which a labeled nucleotide failed to incorporated becomes occupied by an unlabeled nucleotide.
- target polynucleotides may be a specific portion of a genome of a cell, such as an intron, regulatory region, allele, variant or mutation; the whole genome; or any portion therebetween.
- the target polynucleotides may be mRNA, tRNA, rRNA, ribozymes, antisense RNA or RNAi.
- the target polynucleotide may be of any length, such as at least 10 bases, at least 25 bases, at least 50 bases, at least 100 bases, at least 500 bases, at least 1000 bases, or at least 2500 bases.
- the invention is particularly useful in high throughput sequencing of single molecule polynucleotides in which a plurality of target polynucleotides are attached to a solid support in a spatial arrangement such that each polynucleotides is individually optically resolvable.
- each detected incorporated label represents a single polynucleotide
- Nucleotides useful in the invention include both naturally-occurring and modified or non-naturally occurring nucleotides, and include nucleotide analogues.
- a nucleotide according to the invention may be, for example, a ribonucleotide, a deoxyribonucleotide, a modified ribonucleotide, a modified deoxyribonucleotide, a peptide nucleotide, a modified peptide nucleotide or a modified phosphate-sugar backbone nucleotide.
- Many aspects of nucleotides useful in the methods of the invention are subject to manipulation provide and suitable mechanisms for controlling the reaction.
- nucleotide i.e., natural or synthetic dATP, dCTP, dTTP, dGTP or dUTP; a natural or non-natural nucleotide
- dATP dCTP
- dTTP dGTP
- dUTP dUTP
- a natural or non-natural nucleotide will affect the rate or efficiency of the reaction and therefore require consideration in preselecting parameters to produce the desire results.
- Labeled nucleotides of the invention include any nucleotide that has been modified to include a label which is directly or indirectly detectable.
- Such labels include optically-detectable labels such fluorescent labels, including fluorescein, rhodamine, phosphor, polymethadine dye, fluorescent phosphoramidite, texas red, green fluorescent protein, acridine, cyanine, cyanine 5 dye, cyanine 3 dye, 5-(2′-aminoethyl)-aminonaphthalene-1-sulfonic acid (EDANS), BODIPY, ALEXA, or a derivative or modification of any of the foregoing.
- fluorescence resonance energy transfer (FRET) technology is employed to produce a detectable, but quenchable, label.
- FRET may be used in the invention by, for example, modifying the primer to include a FRET donor moiety and using nucleotides labeled with a FRET acceptor moiety.
- the fluorescently labeled nucleotides can be obtained commercially (e.g., from NEN DuPont, Amersham, and BDL). Alternatively, fluorescently labeled nucleotides can also be produced by various techniques, such as those described in Kambara et al., Bio/Techol. (1988) 6:816-821; Smith et al., Nucl. Acid Res. (1985) 13: 2399-2412, and Smith et al., Nature (1986) 321: 674-79.
- the fluorescent dye is preferably linked to the deoxyribose by a linker arm which is easily cleaved by chemical or enzymatic means.
- the length of the linker between the dye and the nucleotide can impact the incorporation rate and efficiency (see Zhu et al., Cytometry (1997) 28, 206).
- nucleotides labeled with any form of detectable label including radioactive labels, chemoluminescent labels, luminescent labels, phosphorescent labels, fluorescence polarization labels, and charge labels.
- any parameter that permits the regulation of the number of labeled nucleotides added to the primer, or the rate at which the nucleotides are incorporated and detected can be controlled or exploited in the practice of the invention.
- Such parameters include, for example, the presence or absence of a label on a nucleotide, the type of label and manner of label attachment; the linker identity and length used to attach the label; the type of nucleotide (including, for example, whether such nucleotide is a dATP, dCTP, dTTP, dGTP or dUTP; a natural or non-natural nucleotide, a nucleotide analogue, or a modified nucleotide); the local sequence immediately 3′ to the addition position; whether the base is the first, second, third, etc. base added; the type of polymerase used; the particular batch characteristics of the polymerase; the processivity of the polymerase; the incorporation rate of the polymerase, and use of polymerase co
- a variety of the conditions of the reaction provide useful mechanisms for controlling either the number of nucleotides incorporated in a single extension reaction or the rates of nucleotide incorporation and detection.
- Such conditions include the “half-life” of the extension cycle (where one half-life is the time taken for at least one incorporation to occur in 50% of the complementary strands); the number of wash cycles (i.e., the number of times a nucleotide is introduced to the reaction then washed out); the number of target nucleic acids in the reaction; and the temperature of the reaction and the reagents used in the reaction.
- the cycle period may also be chosen to permit a certain chance of incorporation of a given number of nucleotides in a complementary strand, and the cycle may be repeated a number of times to analyze the sequence of various numbers of target polynucleotides of varying length.
- nucleotide half-lives for the incorporation reaction are affected by the fact that polymerizing agent may incorporate labeled nucleotides less readily than unlabeled nucleotides.
- FIG. 4 illustrates the statistics of incorporation for a certain embodiment using a Klenow exo-minus polymerizing agent and Cy3- or Cy5-labeled nucleotides. The results show that polymerase may incorporate subsequent labeled nucleotides less readily than a prior labeled nucleotide. The graph of FIG. 4 indicates, for example, that it may take five to ten times longer, resulting in a “stalling” of the incorporation reaction. In other embodiments, the stalling may vary with the use of other labeled nucleotides, other polymerizing agents and various reaction conditions.
- Polymerase stalling is a useful mechanism for controlling incorporation rates in single molecule reactions. As is shown in the Examples below, polymerase stalling is useful to limit incorporation of nucleotides into any given strand in a fairly precise manner. According to the invention, polymerase stalling is useful to limit incorporation to 1 nucleotide per strand per cycle, on average. Given a priori knowledge of the statistics of incorporation, single molecule reactions are controlled to provide a statistical likelihood that 1, sometimes 2, but rarely 3 nucleotides are incorporated in a strand in any given cycle.
- the rate at which polymerase incorporates labeled nucleotides into a complementary strand may be slowed by a factor of about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, or about 15 times compared to that observed with unlabeled nucleotides or compared to that observed for a prior incorporated labeled nucleotide.
- FIGS. 3 and 4 illustrate the results of simulations in which various factors affecting incorporation rates are taken into account.
- the graph of FIG. 4 shows the number of cycles needed with cycle periods of various half-lives, taking into account stalling factors of two (squares), five (triangles), and 10 (crosses), in order to obtain 25 incorporations in over 80% of target strands, with at least a 97% chance of incorporating two or fewer nucleotides per cycle (or a smaller than 3% chance of incorporating three or more nucleotides per cycle).
- FIG. 5 illustrates, if the use of labeled nucleotides slows down the polymerizing agent by a factor of 5, a cycle period of 2.4 half-lives produces over 80% 25-mers in 30 cycles. Based on the teachings of the invention, one of ordinary skill in the art can determine the cycle period required to limit the number incorporations per cycle for a given number of target polynucleotides of a given length.
- the cycle period may be selected to permit about a 70%, about a 75%, about an 80%, about an 85%, about a 90%, about a 95%, about a 96%, about a 97%, about a 98%, and about a 99% chance of incorporation of two or less nucleotides into the complementary strand.
- cycle periods that may be used in embodiments of the invention include, for example, no more than about 5 half-lives, no more than about 4 half-lives, no more than about 3 half-lives, no more than about 2 half-lives, no more than about 1 half-lives, no more than about 0.9 half-lives, no more than about 0.8 half-lives, no more than about 0.7 half-lives, no more than about 0.6 half-lives, no more than about 0.5 half-lives, no more than about 0.4 half-lives, no more than about 0.3 half-lives, and no more than about 0.2 half-lives of the incorporation reactions.
- various cycle periods and number of times the cycles are repeated may be used with various numbers of targets in certain embodiments of the invention. These include, for example, using about 200 target polynucleotides, a period of no more than about 0.6 half-lives and repeating at least about 50 times; using about 200 target polynucleotides, a period of no more than about 0.6 half-lives and repeating at least about 60 times; using about 200 target polynucleotides, a period of no more than about 0.6 half-lives and repeating at least about 70 times; using about 200 target polynucleotides, a period of no more than about 0.8 half-lives and repeating at least about 50 times; using about 200 target polynucleotides, a period of no more than about 0.8 half-lives and repeating at least about 60 times; using about 200 target polynucleotides, a period of no more than about 0.8 half-lives and repeating at least about 70 times; using about 200 target target polynucleot
- the number of times the cycles need to be repeated is also determined based on methods described herein. In general, the number of cycles increases with the length of the sequence to be analyzed and the duration of the half life of nucleotide exposure decreases as the length of sequence to be analyzed becomes longer. Also in general, half lives of nucleotide exposure increase and cycle numbers decrease with greater inhibitory or delaying effects on nucleotide incorporation
- examples of cycle periods and number repeat cycles that may be used in certain embodiments further include a cycle period of no more than about 0.5 half-lives with a stalling factor of about 2, repeated at least about 90 times; a cycle period of no more than about 0.75 half-lives, with a stalling factor of about 2, repeated at least about 75 times; a cycle period of no more than about 1 half-lives, with a stalling factor of about 2, repeated at least about 50 times; a cycle period of no more than about 1.5 half-lives with a stalling factor of about 2 or about 5, repeated at least about 45 times; a cycle period of no more than about 1.75 half-lives, with a stalling factor of about 5, repeated at least about 35 times; a cycle period of no more than about 2 half-lives, with a stalling factor of about 5 or about 10, repeated at least about 35 times; a cycle period of no more than about 2.25 half-lives, with a stalling factor of about 5 or about
- Polymerizing agents useful in the invention include DNA polymerases (such as Taq polymerase, T7 mutant DNA polymerase, Klenow and Sequenase, 9° N or a variant thereof), RNA polymerases, thermostable polymerases, thermodegradable polymerases, and reverse transcriptases.
- DNA polymerases such as Taq polymerase, T7 mutant DNA polymerase, Klenow and Sequenase, 9° N or a variant thereof
- RNA polymerases such as Taq polymerase, T7 mutant DNA polymerase, Klenow and Sequenase, 9° N or a variant thereof
- RNA polymerases such as Taq polymerase, T7 mutant DNA polymerase, Klenow and Sequenase, 9° N or a variant thereof
- thermostable polymerases such as Tet al.
- thermodegradable polymerases such as reverse transcriptases.
- Cofactors of the invention function to inhibit the polymerizing agent, thereby slowing or stopping synthesis activity, permitting the detection of an incorporated labeled nucleotide.
- Cofactors of the invention include any chemical agent or reaction condition that results in the inhibition of the polymerizing agent. Such inhibition may be in whole or in part and may be permanent, temporary or reversible.
- a cofactor may be a label, an antibody, an aptamer, an organic or inorganic small molecule, or a polyanion, or it may comprise a chemical modification to a nucleotide (i.e., a nucleotide analogue may comprise a cofactor).
- a cofactor can be in solution, or it may be attached, either directly or through a linker to a nucleotide, primer, template or polymerase.
- useful cofactor agents include, among others, light sensitive groups such as 6-nitoveratryloxycarbonyl (NVOC), 2-nitobenzyloxycarbonyl (NBOC), ⁇ , ⁇ -dimethyl-dimethoxybenzyloxycarbonyl (DDZ), 5-bromo-7-nitroindolinyl, o-hyrdoxy-2-methyl cinnamoyl, 2-oxymethylene anthraquinone, and t-butyl oxycarbonyl (TBOC).
- NVOC 6-nitoveratryloxycarbonyl
- NBOC 2-nitobenzyloxycarbonyl
- DDZ ⁇ , ⁇ -dimethyl-dimethoxybenzyloxycarbonyl
- TBOC t-butyl oxycarbonyl
- the cofactor may also be the detectable label.
- Labels useful as combined labels/cofactors include larger or bulky dyes.
- the detectable label may comprise a dye having a bulky chemical structure that, once the nucleotide is incorporated into the extending primer, causes a steric hindrance of the polymerizing agent, blocking the polymerizing agent from any further synthesis. Examples of labels that may be useful for this purpose are described in the Example, as well as in Zhu et al., Polynucleotides Res. (1994) 22: 3418-22.
- fluorophore labels that may be used to stall the polymerase include Cy3, Cy5, Cy7, ALEXA647, ALEXA 488, BODIPY 576/589, BODIPY 650/665, BODIPY TR, Nile Blue, Sulfo-IRD700, NN382, R6G, Rho123, tetramethylrhodamine and Rhodamine X.
- the labels are as bulky as Cy5, with molecular weights at least about 1.5 kDa.
- the labels are bulkier than Cy5, having molecular weights of at least about 1.6 kDa, at least about 1.7 kDa, at least about 1.8 kDa, at least about 1.9 kDa, at least about 2.0 kDa at least about 2.5 kDa, or at least about 3.0 kDa.
- Such larger dyes include the following, with corresponding formula weights (in g/mol) in parentheses: Cy5 (534.6); Pyrene (535.6); 6-Carboxyfluorescein (FAM) (537.5); 6-Carboxyfluorescein-DMT (FAM-X (537.5); 5(6) Carboxyfluorescein (FAM) (537.5); 5-Fluorescein (FITC) (537.6); Cy3B (543.0); WellRED D4-PA (544.8); BODIPY 630/650 (545.5); 3′ 6-Carboxyfluorescein (FAM) (569.5); Cy3.5 (576.7); Cascade Blue (580.0); ALEXA Fluor 430 (586.8); Lucifer Yellow (605.5); ALEXA Fluor 532 (608.8); WellRED D2-PA (611.0); Cy5.5 (634.8); DY-630 (634.8); DY-555 (636.2); WellRED D3-PA (645.0); Rhodamine Red-X (65
- the method further comprises inactivating the cofactor, thereby reversing its effect on the polymerizing agent.
- Modes of inactivation depend on the cofactor.
- inactivation can typically be achieved by chemical, enzymatic, photochemical or radiation cleavage of the cofactor from the nucleotide. Cleavage of the cofactor can be achieved if a detachable connection between the nucleotide and the cofactor is used.
- the use of disulfide bonds enables one to disconnect the dye by applying a reducing agent like dithiothreitol (DTT).
- DTT dithiothreitol
- the cofactor is a fluorescent label
- inactivation may comprise adjusting the reaction temperature.
- an antibody that binds to thermostable polymerase at lower temperatures and blocks activity, but is denatured at higher temperatures, thus rendering the polymerase active; or single-stranded aptamers that bind to thermophilic polymerase at lower temperatures but are released at higher temperatures, may be inactivated by increasing the reaction temperature such the cofactor is released but polymerase activity is permitted.
- transient inhibition of the polymerase and the time of exposure to the labeled nucleotide are coordinated such that it is statistically likely that at least one of the labeled nucleotide is incorporated in the primer, but statistically unlikely that more than two of the labeled nucleotide are incorporated.
- the reaction is controlled by inhibiting the polymerase activity such that it is statistically unlikely that more than, for example, one or two nucleotides are incorporated into the same primer strand in the cycle.
- reaction temperature and reagents include reaction temperature and reagents.
- a temperature above or below the temperature required for optimal activity of the polymerizing agent such as a temperature of about 20-70°, would be expected to result in a modulation of the polymerization rate, C.
- This form of inhibition is typically reversible with correction of the reaction temperature, provided that the delta in temperature was insufficient to cause a permanent damage to the polymerase.
- buffer reagents useful in the methods of the invention include a detergent or surfactant, such as Triton®-X 100, or salt and/or ion concentrations that facilitate or inhibit nucleotide incorporation.
- a detergent or surfactant such as Triton®-X 100, or salt and/or ion concentrations that facilitate or inhibit nucleotide incorporation.
- the predetermined point at which a short cycle is stopped is defined, for example, by the occurrence of an event (such as the incorporation of a nucleotide comprising a blocking moiety that prevents further extension of the primer), the lapse of a certain amount of time (such as a specific number of half-lives), or the achievement of a statistically-significant datapoint (such as a period at which a statistically significant probability of two or less nucleotides have been incorporated).
- the predetermined period of time is coordinated with an amount of polymerization inhibition such that, on average, a certain number of labeled nucleotides are added to the primer.
- the number of incorporated labeled nucleotides is, on average, 0, 1 or 2, but almost never more than 3.
- the time period of exposure is defined in terms of statistical significance.
- the time period may be that which is statistically insufficient for incorporation of more nucleotides than are resolvable by a detection system used to detect incorporation of the nucleotide into the primer.
- the time period that is statistically insufficient for incorporation of a greater number of nucleotides that are individually optically resolvable during a predetermined detection period i.e., a period of time during which the incorporated nucleotides are detected).
- the reaction may be stopped by washing or flushing out the nucleotides that remain unincorporated and/or washing or flushing out polymerization agent. Further, many aspects of the repeated cycles may be automated, for example, using microfluidics for washing nucleotides to sites of anchored target polynucleotides, and washing out unincorporated nucleotides to halt each cycle.
- Primers are synthesized from nucleoside triphosphates by known automated oligonucleotide synthetic techniques, e.g., via standard phosphoramidite technology utilizing a nucleic acid synthesizer, such as the ABI3700 (Applied Biosystems, Foster City, Calif.).
- the oligonucleotides are prepared as duplexes with a complementary strand, however, only the 5′ terminus of the oligonucleotide proper (and not its complement) is biotinylated.
- Double stranded target nucleic acids are blunt-end ligated to the oligonucleotides in solution using, for example, T4 ligase.
- the single strand having a 5′ biotinylated terminus of the oligonucleotide duplex permits the blunt-end ligation on only on end of the duplex.
- the solution-phase reaction is performed in the presence of an excess amount of oligonucleotide to prohibit the formation of concantamers and circular ligation products of the target nucleic acids.
- a plurality of chimeric polynucleotide duplexes result. Chimeric polynucleotides are separated from unbound oligonucleotides based upon size and reduced to single strands by subjecting them to a temperature that destabilizes the hydrogen bonds.
- a solid support comprising reaction chambers having a fused silica surface is sonicated in 2% MICRO-90 soap (Cole-Parmer, Vernon Hills, Ill.) for 20 minutes and then cleaned by immersion in boiling RCA solution (6:4:1 high-purity H 2 O/30% NH 4 OH/30% H 2 O 2 ) for 1 hour. It is then immersed alternately in polyallylamine (positively charged) and polyacrylic acid (negatively charged; both from Aldrich) at 2 mg/ml and pH 8 for 10 minutes each and washed intensively with distilled water in between.
- the slides are incubated with 5 mM biotin-amine reagent (Biotin-EZ-Link, Pierce) for 10 minutes in the presence of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC, Sigma) in MES buffer, followed by incubation with Streptavidin Plus (Prozyme, San Leandro, Calif.) at 0.1 mg/ml for 15 minutes in Tris buffer.
- the biotinylated single-stranded chimeric polynucleotides are deposited via ink jet printing onto the streptavidin-coated chamber surface at 10 pM for 10 minutes in Tris buffer that contain 100 mM MgCl 2 .
- the experiments are performed on an upright microscope (BH-2, Olympus, Melville, N.Y.) equipped with total internal reflection (TIR) illumination, such as the BH-2 microscope from Olympus (Melville, N.Y.).
- TIR total internal reflection
- Two laser beams, 635 (Coherent, Santa Clara, Calif.) and 532 nm (Brimrose, Baltimore), with nominal powers of 8 and 10 mW, respectively, are circularly polarized by quarter-wave plates and undergo TIR in a dove prism (Edmund Scientific, Barrington, N.J.).
- the prism is optically coupled to the fused silica bottom (Esco, Oak Ridge, N.J.) of the reaction chambers so that evanescent waves illuminated up to 150 nm above the surface of the fused silica.
- An objective (DPlanApo, 100 UV 1.3oil, Olympus) collects the fluorescence signal through the top plastic cover of the chamber, which is deflected by the objective to ⁇ 40 ⁇ m from the silica surface.
- An image splitter (Optical Insights, Santa Fe, N.M.) directs the light through two bandpass filters (630dcxr, HQ585/80, HQ690/60; Chroma Technology, Brattleboro, Vt.) to an intensified charge-coupled device (I-PentaMAX; Roper Scientific, Trenton, N.J.), which records adjacent images of a 120- ⁇ 60- ⁇ m section of the surface in two colors.
- chimeric polynucleotides i.e., the polynucleotide portion added to the bound oligonucleotides is different at least one location
- all four labeled dNTPs initially are cycled. The result is the addition of at least one donor fluorophore to each chimeric strand.
- the number of initial incorporations containing the donor fluorophore is limited by either limiting the reaction time (i.e., the time of exposure to donor-labeled nucleotides), by polymerase stalling, or both in combination.
- the inventors have shown that base-addition reactions are regulated by controlling reaction conditions. For example, incorporations can be limited to 1 or 2 at a time by causing polymerase to stall after the addition of a first base.
- One way in which this is accomplished is by attaching a dye to the first added base that either chemically or sterically interferes with the efficiency of incorporation of a second base.
- a computer model was constructed using Visual Basic (v.
- the model utilizes several variables that are thought to be the most significant factors affecting the rate of base addition.
- the number of half-lives until dNTPs are flushed is a measure of the amount of time that a template-dependent system is exposed to dNTPs in solution. The more rapidly dNTPs are removed from the template, the lower will be the incorporation rate.
- the number of wash cycles does not affect incorporation in any given cycle, but affects the number bases ultimately added to the extending primer.
- the number of strands to be analyzed is a variable of significance when there is not an excess of dNTPs in the reaction.
- FIG. 2 is a screenshot showing the inputs used in the model.
- the model demonstrates that, by controlling reaction conditions, one can precisely control the number of bases that are added to an extending primer in any given cycle of incorporation. For example, as shown in FIG. 7 , at a constant rate of inhibition of second base incorporation (i.e., the inhibitory effect of incorporation of a second base given the presence of a first base), the amount of time that dNTPs are exposed to template in the presence of polymerase determines the number of bases that are statistically likely to be incorporated in any given cycle (a cycle being defined as one round of exposure of template to dNTPs and washing of unbound dNTP from the reaction mixture). As shown in FIG.
- FIG. 2 shows schematically the process of FRET-based, template-dependent nucleotide addition as described in this example.
- FRET fluorescence resonance spectroscopy
- donor follows the extending primer as new nucleotides bearing acceptor fluorophores are added.
- a nucleotide binding protein e.g., DNA binding protein
- the DNA binding protein is spaced at intervals (e.g., about 5 nm or less) to allow FRET.
- FRET FRET to conduct single molecule sequencing using the devices and methods taught in the application.
- incorporated nucleotides are detected by virtue of their optical emissions after sample washing.
- Primers are hybridized to the primer attachment site of bound chimeric polynucleotides Reactions are conducted in a solution comprising Klenow fragment Exo-minus polymerase (New England Biolabs) at 10 nM (100 units/nil) and a labeled nucleotide triphosphate in EcoPol reaction buffer (New England Biolabs). Sequencing reactions takes place in a stepwise fashion. First, 0.2 ⁇ M dUTP-Cy3 and polymerase are introduced to support-bound chimeric polynucleotides, incubated for 6 to 15 minutes, and washed out.
- Images of the surface are then analyzed for primer-incorporated U-Cy5. Typically, eight exposures of 0.5 seconds each are taken in each field of view in order to compensate for possible intermittency (e.g., blinking) in fluorophore emission.
- Software is employed to analyze the locations and intensities of fluorescence objects in the intensified charge-coupled device pictures. Fluorescent images acquired in the WinView32 interface (Roper Scientific, Princeton, N.J.) are analyzed using ImagePro Plus software (Media Cybernetics, Silver Springs, Md.). Essentially, the software is programmed to perform spot-finding in a predefined image field using user-defined size and intensity filters.
- the program then assigns grid coordinates to each identified spot, and normalizes the intensity of spot fluorescence with respect to background across multiple image frames. From those data, specific incorporated nucleotides are identified.
- the type of image analysis software employed to analyze fluorescent images is immaterial as long as it is capable of being programmed to discriminate a desired signal over background.
- the programming of commercial software packages for specific image analysis tasks is known to those of ordinary skill in the art. If U-Cy5 is not incorporated, the substrate is washed, and the process is repeated with dGTP-Cy5, dATP-Cy5, and dCTP-Cy5 until incorporation is observed. The label attached to any incorporated nucleotide is neutralized, and the process is repeated. To reduce bleaching of the fluorescence dyes, an oxygen scavenging system can be used during all green illumination periods, with the exception of the bleaching of the primer tag.
- the above protocol is performed sequentially in the presence of a single species of labeled dATP, dGTP, dCTP or dUTP.
- a first sequence can be compiled that is based upon the sequential incorporation of the nucleotides into the extended primer.
- the first compiled sequence is representative of the complement of the template.
- the sequence of the template can be easily determined by compiling a second sequence that is complementary to the first sequence. Because the sequence of the oligonucleotide is known, those nucleotides can be excluded from the second sequence to produce a resultant sequence that is representative of the target template.
- FIG. 2 illustrates the advantage of short-cycle sequencing with respect to avoiding long homopolymer reads.
- FIG. 2 a illustrates a simulated analysis of 10 target polynucleotides using non-short-cycle sequencing (Example 2a)
- FIG. 2 b illustrates a simulated analysis of the same number of target polynucleotides using short-cycle sequencing (Example 2b).
- the simulations were performed as follows: an Excel spreadsheet was opened and “Customize . . . ” selected from the “Tools” menu of the Excel toolbar. The “Commands” tab was selected and, after scrolling down, “Macros” was clicked. The “smiley face” that appeared in the right panel was dragged to the toolbars on top of the spreadsheet. The “Customize” box was closed and the “smiley face” clicked once. From the list of subroutines that appeared, “ThisWorkbook.Main_Line.” was selected. The program was run by clicking again on the “smiley face.” A copy of the source code for the Excel simulation is provided below.
- Input values were then entered into the tabbed sheet called “In Out.” There were three input values:
- the first input value corresponded to the period of time allowed for incorporation reactions of provided nucleotides into the growing complementary strands of the polynucleotides to be analyzed. This period was conveniently measured in half-lives of the incorporation reaction itself. Each cycle of incorporation was simulatedly stalled after a period of time, representing, for example, the time when unincorporated nucleotides would be flushed out or the incorporation reactions otherwise stalled.
- the second input value corresponds to the number of times each cycle of incorporation was repeated. That is, the number of times the steps of providing nucleotides, allowing incorporation reactions into the complementary strands in the presence of polymerizing agent, and then stopping the incorporations are repeated.
- the nucleotides were simulatedly provided as a wash of each of dATPs, dGTPs, dTTPs, and dCTPs. The program then recorded which nucleotides were incorporated, corresponding to a detection step of detecting incorporation.
- the third input value corresponds to number of strands of target polynucleotides to by analyzed in the simulation.
- the program allowed up to 1100 target polynucleotide molecules to be analyzed in a given simulation.
- the program After the program was started, as described above, the program first generated the inputted number of strands composed of random sequences. The program then simulated hybridization and polymerization of the correct base of each incorporation reaction, based on the generated sequence of the target polynucleotide templates. The program continued these simulated reactions for the allowed amount of simulated time, determined by the inputted number of half-lives. Statistics of the simulation were then computed and reported, including the longest strand, the shortest strand, and the average length of all strands, as well as the fraction of strands extended by at least 25 nucleotide incorporations, as discussed in more detail below.
- Example 2a the input values used were a cycle period of 10 half-lives, 12 repeats of the cycle, and 10 target polynucleotide strands.
- FIG. 2 a illustrates the results obtained. Homopolymers stretches which occurred in the same simulated complementary strand are highlighted in magenta wherever 2 nucleotides of the same base type were incorporated in a row, and in cyan wherever more than two nucleotides of the same base type were incorporated in a row.
- FIG. 2 a illustrates that the output values included the longest extended complementary strand obtained during the simulation (Longest extension in the ensemble of molecules); the shorted extended complementary strand obtained during the simulation (Shortest extension in the ensemble of molecules); and the average extension. These numbers represent the greatest number of incorporations into any of the 10 simulatedly growing complementary strands, the smallest number of incorporations for any of the 10, and the average number of incorporations for the 10.
- FIG. 2 a indicates that the values obtained for Example 2a were 37 incorporations in the longest extension, 25 in the shortest, and 30.00 as the average number of incorporations.
- the output values also provided information on the number of incorporations that occurred in each of growing complementary strands during each cycle period of the simulation.
- FIG. 2 a indicates that for the input values of Example 2a, the percentage of growing stands extended by two or more nucleotides in a homopolymer stretch was 100.0%; and the percentage of growing strands extended by three or more nucleotides in a homopolymer stretch was 60.0%. That is, using a cycle period of 10 half-lives resulted in only 40% of the complementary strands being extended by two or less nucleotides in a homopolymer stretch per cycle of incorporation.
- output values also indicated the total number of incorporations for each of the growing strands for the total number of repeated cycles. This represents the length of the sequence of target polynucleotide analyzed.
- FIG. 2 a illustrates that in Example 2a, 100.0% of the 10 target polynucleotides of the simulation were extended by at least 25 incorporated nucleotides. This illustrates that using a cycle period of 10 half-lives, and repeating the cycles 12 times, allowed analysis of a 25 base sequence of 10 target polynucleotides.
- Example 2b the input values used were a cycle period of 0.8 half-lives, 60 repeats of the cycle, and 10 target polynucleotide strands.
- FIG. 2 b illustrates the results obtained. Homopolymers stretches which occurred in the same simulated complementary strand are highlighted in magenta wherever 2 nucleotides of the same base type were incorporated in a row, and in cyan wherever more than two nucleotides of the same base type were incorporated in a row.
- FIG. 2 b illustrates that the output values included the longest extended complementary strand obtained during the simulation (longest extension in the ensemble of molecules); the shortest extended complementary strand obtained during the simulation (shortest extension in the ensemble of molecules); and the average extension. These numbers represent the greatest number of incorporations into any of the 10 simulatedly growing complementary strands, the smallest number of incorporations for any of the 10, and the average number of incorporations for the 10.
- FIG. 2 b indicates that the values obtained for Example 2b were 37 incorporations in the longest extension, 26 in the shortest, and 32.00 as the average number of incorporations.
- the output values also provided information on the number of incorporations that occurred in each of growing complementary strands during each cycle period of the simulation.
- FIG. 2 b indicates that for the input values of Example 2b, the percentage of growing stands extended by two or more nucleotides in a homopolymer stretch was 80.0%; and the percentage of growing strands extended by three or more nucleotides in a homopolymer stretch was 10.0%. That is, using a cycle period of 0.8 half-lives resulted in 90% of the complementary strands being extended by two or less nucleotides per cycle of incorporation.
- Output values also indicated the total number of incorporations for each of the growing strands for the total number of repeated cycles. As in Example 2a, this represents the length of the sequence of target polynucleotide analyzed.
- FIG. 2 b illustrates that in Example 2b, 100.0% of the 10 target polynucleotides of the simulation were again extended by at least 25 incorporated nucleotides. This illustrates that using a cycle period of 0.8 half-lives, and repeating the cycles 60 times, allowed analysis of a 25 base sequence of 10 target polynucleotides.
- Comparing Examples 2a and 2b also indicated that a greater number of repeated cycles were needed to analyze a given length of sequence when using shorter cycles. That is, the 10 half-lives cycle was repeated 12 times to result in 100.0% of the 10 complementary strands being extended by at least 25 nucleotides, whereas the 0.8 half-lives cycle was repeated 60 times to obtain this same result and thereby analyze the 25 nucleotides sequence.
- repeated cycles may be automated, for example, using micro fluidics for washing nucleotides to sites of anchored target polynucleotides, and washing out unincorporated nucleotides to halt each cycle.
- FIG. 2 illustrates yet another simulated analysis of a number of target polynucleotides using short-cycle sequencing. The simulation was run using the program described in Examples 2a and 2b but using a larger number of target polynucleotides.
- FIG. 2 illustrates the results obtained. Homopolymers stretches which occurred in the same simulated complementary strand are highlighted in magenta wherever nucleotides of the same base type were incorporated in a row, and in cyan wherever more than two nucleotides of the same base type were incorporated in a row.
- the output values obtained were 48 incorporations in the longest extended complementary strand, 20 in the shortest, and 32.00 as the average number of incorporations for the 200 simulatedly extended complementary strands.
- the percentage of growing stands extended by two or more nucleotides in a homopolymer stretch was 78.5%; and the percentage of growing strands extended by three or more nucleotides in a homopolymer stretch was 4.0%. That is, using a cycle period of 0.8 half-lives resulted in 96.0% of the complementary strands being extended by two or less nucleotides in a homopolymer stretch per cycle of incorporation. Moreover, 95.5% of the 200 target polynucleotides of the simulation were extended by at least 25 incorporated nucleotides, while 100% were extended by at least 20 nucleotides. This illustrated that using a cycle period of 0.8 half-lives, and repeating the cycles 60 times, allows analysis of a 20 base sequence of 200 target polynucleotides.
- This example demonstrates a method according to the invention in which a single nucleotide in a position in a nucleic acid sequence is identified.
- a template-bound primer is sequentially exposed first to a labeled nucleotide and then to an unlabeled nucleotide of the same type under conditions and in the presence of reagents that allow template-dependent primer extension.
- the template is analyzed in order to determine whether the first nucleotide is incorporated in the primer at the first position or not. If not, then the sequential exposure to labeled and unlabeled nucleotides is repeated using another type of nucleotide until one such nucleotide is determined to have incorporated at the first position. Once an incorporated nucleotide is determined, the identity of the nucleotide in the position in the nucleic acid sequence is identified as the complementary nucleotide.
- a nucleic acid primer is hybridized to a target nucleic acid at a primer binding site in the target.
- the primer comprises a donor fluorophore.
- the hybridized primer is exposed to a first nucleotide comprising an acceptor fluorophore comprising a blocking moiety that, when incorporated into the primer, prevents further polymerization of the primer.
- the presence or absence of fluorescent emission from each of the donor and the acceptor is determined.
- a nucleotide that has been incorporated into the primer via complementary base pairing with the target is identified by the presence of fluorescent emission from the acceptor, and a sequence placeholder is identified as the absence of fluorescent emission from the donor and the acceptor.
- a sequence of the target nucleic acid is complied based upon the sequence of the incorporated nucleotides and the placeholders.
Abstract
Description
- This application claims the benefit of U.S. Provisional Application Nos. 60/546,277, filed on Feb. 19, 2004, 60/547,611, filed on Feb. 24, 2004, and 60/519,862, filed on Nov. 11, 2003.
- The invention relates to methods for sequencing a polynucleotide, and more particularly, to methods for high throughput single molecule sequencing of target polynucleotides.
- Completion of the human genome has paved the way for important insights into biologic structure and function. Knowledge of the human genome has given rise to inquiry into individual differences, as well as differences within an individual, as the basis for differences in biological function and dysfunction. For example, single nucleotide differences between individuals, called single nucleotide polymorphisms (SNPs), are responsible for dramatic phenotypic differences. Those differences can be outward expressions of phenotype or can involve the likelihood that an individual will get a specific disease or how that individual will respond to treatment. Moreover, subtle genomic changes have been shown to be responsible for the manifestation of genetic diseases, such as cancer. A true understanding of the complexities in either normal or abnormal function will require large amounts of specific sequence information.
- An understanding of cancer also requires an understanding of genomic sequence complexity. Cancer is a disease that is rooted in heterogeneous genomic instability. Most cancers develop from a series of genomic changes, some subtle and some significant, that occur in a small subpopulation of cells. Knowledge of the sequence variations that lead to cancer will lead to an understanding of the etiology of the disease, as well as ways to treat and prevent it. An essential first step in understanding genomic complexity is the ability to perform high-resolution sequencing.
- Various approaches to nucleic acid sequencing exist. One conventional way to do bulk sequencing is by chain termination and gel separation, essentially as described by Sanger et al., Proc Natl Acad Sci USA, 74(12): 5463-67 (1977). That method relies on the generation of a mixed population of nucleic acid fragments representing terminations at each base in a sequence. The fragments are then run on an electrophoretic gel and the sequence is revealed by the order of fragments in the gel. Another conventional bulk sequencing method relies on chemical degradation of nucleic acid fragments. See, Maxam et al., Proc. Natl. Acad. Sci., 74: 560-564 (1977). Finally, methods have been developed based upon sequencing by hybridization. See, e.g., Drmanac, et al., Nature Biotech., 16: 54-58 (1998). Bulk techniques, such as those described above, cannot effectively detect single nucleotide differences between samples, and are not useful for comparative whole genome sequencing. Single molecule techniques are necessary for high-resolution detection of sequence differences.
- There have been several recent reports of sequencing using single molecule techniques. Most conventional techniques have proposed incorporation of fluorescently-labeled nucleotides in a template-dependent manner. A fundamental problem with conventional single molecule techniques is that the sequencing reactions are run to completion. For purposes of single molecule chemistry, this typically means that template is exposed to nucleotides for incorporation for about 10 half lives. This gives rise to problems in the ability to resolve single nucleotides as they incorporate in the growing primer strand. The resolution problem becomes extreme in the situation in which the template comprises a homopolymer region. Such a region is a continuous sequence consisting of the same nucleotide species. When optical signaling is used as the detection means, conventional optics are able to reliably distinguish one from two identical bases, and sometimes two from three, but rarely more than three. Thus, single molecule sequencing using fluorescent labels in a homopolymer region typically results in a signal that does not allow accurate determination of the number of bases in the region.
- One method that has been developed in order to address the homopolymer issue provides for the use of nucleotide analogues that have a modification at the 3′ carbon of the sugar that reversibly blocks the hydroxyl group at that position. The added nucleotide is detected by virtue of a label that has been incorporated into the 3′ blocking group. Following detection, the blocking group is cleaved, typically, by photochemical means to expose a free hydroxyl group that is available for base addition during the next cycle.
- However, techniques utilizing 3′ blocking are prone to errors and inefficiencies. For example, those methods require excessive reagents, including numerous primers complementary to at least a portion of the target nucleic acids and differentially-labeled nucleotide analogues. They also require additional steps, such as cleaving the blocking group and differentiating between the various nucleotide analogues incorporated into the primer. As such, those methods have only limited usefulness.
- Need therefore exists for more effective and efficient methods and devices for single molecule nucleic acid sequencing.
- The invention provides methods for high throughput single molecule sequencing. In particular, the invention provides methods for controlling at least one parameter of a nucleotide extension reaction in order to regulate the rate at which nucleotides are added to a primer. The invention provides several ways of controlling nucleic acid sequence-by-synthesis reactions in order to increase the resolution and reliability of single molecule sequencing. Methods of the invention solve the problems that imaging systems have in accurately resolving a sequence at the single-molecule level. In particular, methods of the invention solve the problem of determining the number of nucleotides in a homopolymer stretch.
- Methods of the invention generally contemplate terminating sequence-by-synthesis reactions prior to completion in order to obtain increased resolution of individual nucleotides in a sequence. Fundamentally, this requires exposing nucleotides to a mixture comprising a template, a primer, and a polymerase under conditions sufficient for only limited primer extension. Reactions are conducted under conditions such that it is statistically unlikely that more than 1 or 2 nucleotides are added to a growing primer strand in any given incorporation cycle. An incorporation cycle comprises exposure of a template/primer to nucleotides directed at the base immediately downstream of the primer (this may be all four conventional nucleotides or analogs if the base is not known) and washing unhybridized nucleotide.
- Nucleotide addition in a sequence-by-synthesis reaction is a stochastic process. As in any chemical reaction, nucleotide addition obeys the laws of probability. Methods of the invention are concerned with controlling the rate of nucleotide addition on a per-cycle basis. That is, the invention teaches ways to control the rate of nucleotide addition within an extension cycle given the stochastic nature of the extension reaction itself. Methods of the invention are intended to control reaction rates within the variance that is inherent in a reaction that is fundamentally stochastic. Thus, the ability to control, according to the invention, base addition reactions such that, on average, no more than two bases are added in any cycle takes into account the inherent statistics of the reactions.
- The invention thus teaches polynucleotide sequence analysis using short cycle chemistry. One embodiment of the invention provides methods for slowing or reversibly inhibiting the activity of polymerase during a sequencing-by-synthesis reaction. Other methods teach altering the time of exposure of nucleotides to the template-primer complex. Still other methods teach the use of physical blockers that temporarily halt or slow polymerase activity and/or nucleotide addition. In general, any component of the reaction that permits regulation of the number of labeled nucleotides added to the primer per cycle, or the rate at which the nucleotides are incorporated and detected per cycle is useful in methods of the invention. Additional components include, but are not limited to, the presence or absence of a label on a nucleotide, the type of label and manner of attaching the label; the linker identity and length used to attach the label; the type of nucleotide (including, for example, whether such nucleotide is a dCTP, dTTP, dGTP or dUTP; a natural or non-natural nucleotide, a nucleotide analogue, or a modified nucleotide); the “half-life” of the extension cycle (where one half-life is the time taken for at least one incorporation to occur in 50% of the complementary strands); the local sequence immediately 3′ to the addition position; whether such base is the first, second, third, etc. base added; the type of polymerase used; the particular batch characteristics of the polymerase; the processivity of the polymerase; the incorporation rate of the polymerase; the number of wash cycles (i.e., the number of times a nucleotide is introduced to the reaction then washed out); the number of target nucleic acids in the reaction; the temperature of the reaction and the reagents used in the reaction.
- In a preferred embodiment of the invention, a nucleic acid template is exposed to a primer capable of hybridizing to the template and a polymerase capable of catalyzing nucleotide addition to the primer. A labeled nucleotide is introduced for a period of time that is statistically insufficient for incorporation of more than about 2 nucleotides per cycle. Nucleotide exposure may also be coordinated with polymerization inhibition such that, on average, 0, 1, or 2 labeled nucleotides are added to the primer, but that 3 labeled nucleotides are almost never added to the primer in each cycle. Ideally, the exposure time, during which labeled nucleotides are exposed to the template-primer complex, is statistically insufficient for incorporation of more nucleotides than are resolvable by a detection system used to detect incorporation.
- The invention also contemplates performing a plurality of base incorporation cycles. Each cycle comprises exposing a template nucleic acid to a labeled nucleotide that is not a chain-terminating nucleotide. The labeled nucleotide is incorporated into a primer hybridized to the template nucleic acid if the nucleotide is capable of hybridizing to the template nucleotide immediately upstream of the primer and there is about a 99% probability that two or fewer of said nucleotides are incorporated into the same primer strand per cycle. Incorporated nucleotides are then identified.
- Methods of the invention also make use of differential base incorporation rates in order to control overall reaction rates. For example, the rate of incorporation is lower for a second nucleotide given incorporation of a prior nucleotide immediately upstream of the second. This effect is magnified if the first nucleotide comprises a label or other group that hinders processivity of the polymerase. By determining an approximate reduction in the rate of incorporation of the second nucleotide, one can regulated the time of exposure of a sample to a second labeled nucleotide such that the time is statistically insufficient for incorporation of more nucleotides than are resolvable by a detection system used to detect incorporation of the nucleotide into the primer.
- The invention may also be conducted using a plurality of primer extension cycles, wherein each cycle comprises exposing a target nucleic acid to a primer capable of hybridizing to the target, thereby forming a primed target; exposing the primed target to a labeled nucleic acid in the presence of a nucleic acid polymerase, coordinating transient inhibition of the polymerase and time of exposure to the labeled nucleotide such that it is statistically likely that at least one of said labeled nucleic acid is incorporated in the primer, but statistically unlikely that more than two of the labeled nucleotide are incorporated in the primer.
- According to another embodiment, methods of the invention comprise conducting a cycle of template-dependent nucleic acid primer extension in the presence of a polymerase and a labeled nucleotide; inhibiting polymerase activity such that it is statistically unlikely that more than about 2 nucleotides are incorporated into the same primer strand in the cycle; washing unincorporated labeled nucleotide away from the template; detecting any incorporation of the labeled nucleotide; neutralizing label in any incorporated labeled nucleotide; removing the inhibition; repeating the foregoing steps; and compiling a sequence based upon the sequence of nucleotides incorporated into the primer.
- In another embodiment, the invention provides a method comprising exposing a nucleic acid template to a primer capable of hybridizing to a portion of the template in order to form a template/primer complex reaction mixture; adding a labeled nucleotide in the presence of a polymerase to the mixture under conditions that promote incorporation of the nucleotide into the primer if the nucleotide is complementary to a nucleotide in the template that is downstream of said primer; coordinating removal of the labeled nucleotide and inhibition of the polymerase so that no more than about 2 nucleotides are incorporated into the same primer; identifying labeled nucleotide that has been incorporated into said primer; repeating the foregoing steps at least once; and determining a sequence of the template based upon the order of the nucleotides incorporated into the primer.
- According to another embodiment, the method comprises exposing a template nucleic acid to a primer capable of hybridizing to a portion of the template upstream of a region of the template to be sequenced; introducing a labeled nucleic acid and a polymerase to the template under conditions wherein the labeled nucleic acid will be incorporated in the primer if the labeled nucleic acid is capable of hybridizing with base downstream of the primer; and controlling the rate of the incorporation by limiting the time of exposure of the labeled nucleic acid to the template or by inhibiting the polymerase at a predefined time after exposure of the template to the labeled nucleotide; detecting incorporation of the labeled nucleotide into the primer; and identifying the nucleotide in the template as the complement of labeled nucleotide incorporated into the primer.
- In yet another embodiment, methods of the invention comprise exposing a target polynucleotide to a primer capable of hybridizing to the polynucleotide, extending the primer in the presence of a polymerizing agent and one or more extendible nucleotides, each comprising a detectable label. The polymerizing agent is exposed to a cofactor (i.e., any agent that decreases or halts polymerase activity), and the incorporation of label is detected. The steps of extending the primer and exposing the polymerizing agent to a cofactor may be performed simultaneously, or may be performed in separate steps. In one embodiment, the method further comprises inactivating the cofactor, thereby reversing its effect on the polymerizing agent. Modes of inactivation depend on the cofactor. For example, where the cofactor is attached to the nucleotide, inactivation can typically be achieved by cleaving the cofactor from the nucleotide.
- Methods of the invention also address the problem of reduced detection due to a failure of some strands in a given cycle to incorporate labeled nucleotide. In each incorporation cycle, a certain number of strands fail to incorporate a nucleotide that should be incorporated based upon its ability to hybridize to a nucleotide present in the template. The strands that fail to incorporate a nucleotide in a cycle will not be prepared to incorporate a nucleotide in the next cycle (unless it happens to be the same as the unincorporated nucleotide, in which case the strand will still lag behind unless both nucleotides are incorporated in the same cycle). Essentially, this situation results in the strands that failed to incorporate being unavailable for subsequent polymerase-catalyzed additions to the primer. That, in turn, leads to fewer strands available for base addition in each successive cycle (assuming the non-incorporation occurs in all or most cycles). The invention overcomes this problem by exposing a template/primer complex to a labeled nucleotide that is capable of hybridizing to the template nucleotide immediately downstream of the primer. After removing unbound labeled nucleotide, the sample is exposed to unlabeled nucleotide, preferably in excess, of the same species. The unlabeled nucleotide “fills in” the positions in which hybridization of the labeled nucleotide did not occur. That functions to increase the number of strands that are available for participation in the next round. The effect is to increase resolution in subsequent rounds over background. In a preferred embodiment, the labeled nucleotide comprises a label that impedes the ability of polymerase to add a downstream nucleotide, thus temporarily halting the synthesis reaction until unlabeled nucleotide can be added, at which point polymerase inhibition is removed and t he next incorporation cycle is conducted
- One feature of this embodiment is that a sequence is compiled based upon the incorporation data, while allowing maximum strand participation in each cycle. Thus, methods of the invention are useful for identifying placeholders in some strands in a population of strands being sequenced. As long as there are no more than two consecutive placeholders in any one strand, the invention has a high tolerance for placeholders with little or no effect on the ultimate sequence determination.
- Methods of the invention are also useful for identifying a single nucleotide in a nucleic acid sequence. The method comprises the steps of sequentially exposing a template-bound primer to a labeled nucleotide and an unlabeled nucleotide of the same type in the presence of a polymerase under conditions that allow template-dependent primer extension; determining whether the first nucleotide is incorporated in the primer at a first position; repeating the sequentially exposing step using subsequent labeled and unlabeled nucleotides until a nucleotide is identified at the first position.
- Identification of nucleotides in a sequence can be accomplished according to the invention using fluorescence resonance energy transfer (FRET). Single pair FRET (spFRET) is a good mechanism for increasing signal-to-noise in single molecule sequencing. Generally, a FRET donor (e.g., cyanine-3) is placed on the primer, on the polymerase, or on a previously incorporated nucleotide. The primer/template complex then is exposed to a nucleotide comprising a FRET acceptor (e.g., cyanine-5). If the nucleotide is incorporated, the acceptor is activated and emits detectable radiation, while the donor goes dark. That is the indication that a nucleotide has been incorporated. The nucleotide is identified based upon knowledge of which nucleotide species contained the acceptor. The invention also provides methods for identifying a placeholder in a nucleic acid sequence using FRET. A nucleic acid primer is hybridized to a target nucleic acid at a primer binding site in the target. The primer comprises a donor fluorophore. The hybridized primer is exposed to a first nucleotide comprising an acceptor fluorophore that, when incorporated into the primer, prevents further polymerization of the primer. Whether there is fluorescent emission from the donor and the acceptor is determined, and a placeholder in the nucleic acid sequence is identified as the absence of emission in both the donor and the acceptor.
- In another embodiment, the method comprises hybridizing a nucleic acid primer comprising a donor fluorophore to a target nucleic acid at a primer binding site in the target; exposing the hybridized primer to a first nucleotide comprising an acceptor fluorophore that, when incorporated into the primer, prevents further polymerization of the primer; detecting the presence or absence of fluorescent emission from each of the donor and the acceptor; identifying a nucleotide that has been incorporated into the primer via complementary base pairing with the target as the presence of fluorescent emission from the acceptor; identifying a sequence placeholder as the absence of fluorescent emission from the donor and the acceptor; and repeating the exposing, detecting, and each of the identifying steps, thereby to compile a sequence of the target nucleic acid based upon the sequence of the incorporated nucleotides and the placeholders.
- The invention is useful in sequencing any form of polynucleotides, such as double-stranded DNA, single-stranded DNA, single-stranded DNA hairpins, DNA/RNA hybrids, RNAs with a recognition site for binding of the polymerizing agent, and RNA hairpins. The invention is particularly useful in high throughput sequencing of single molecule polynucleotides in which a plurality of target polynucleotides are attached to a solid support in a spatial arrangement such that each polynucleotides is individually optically resolvable. According to the invention, each detected incorporated label represents a single polynucleotide.
- A detailed description of the certain embodiments of the invention is provided below. Other embodiments of the invention are apparent upon review of the detailed description that follows.
- The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.
-
FIG. 1 shows asynchronous single molecule sequencing. -
FIG. 2 are screenshots showing data from short cycle sequencing with long homopolymer regions.FIG. 2 a shows full cycle sequencing used to analyze 10 target polynucleotides in a simulated synthesis of their complementary strands using cycle periods of 10 half-lives and repeating the wash cycles 12 times.FIG. 2 b shows a short cycle sequencing to analyze 10 target polynucleotides by simulating the synthesis of their complementary strands using short cycle periods of 0.8 half-life periods and repeating the wash cycles 60 times. -
FIG. 3 shows a short cycle embodiment for analyzing 200 target polynucleotides in a simulated synthesis of their complementary strands using short cycle periods of 0.8 half-life periods and repeating the wash cycles 60 times. -
FIG. 4 shows a statistical analysis of incorporation, showing that polymerizing agent may incorporate repeat labeled nucleotides less readily than the first labeled nucleotide. -
FIG. 5 shows a simulation showing the effect of decreasing the activity rate of the polymerizing agent and lengthening half-lives on the cycle period. -
FIG. 6 shows the number of cycles needed with cycle periods of various half-lives taking into account stalling factors of two (squares), five (triangles) and 10 (crosses), in order to obtain over 25 incorporations in over 80% of target homopolymers, with at least a 97% chance of incorporating two or less nucleotides per cycle (or a smaller than 3% chance of incorporating more than 2 nucleotides per cycle). -
FIG. 7 is a series of screenshots showing the effects of altering reaction conditions on the incorporation of nucleotides in a single molecule sequencing by synthesis reaction. - The invention provides methods for high throughput single molecule sequencing. According to the invention, one or more parameters of a sequencing-by-synthesis reaction are preselected such that the incorporation of, preferably, a single nucleotide on a primed target template is optically detectable. In one embodiment, the preselected parameters regulate the rate at which the nucleotides are incorporated, and the rate at which the incorporated nucleotides are detected. According to this embodiment, the nucleotides are individually detected either as they are incorporated or shortly thereafter, essentially in “real-time. In another embodiment, the preselected parameters permit the regulation of the number of nucleotides incorporated during a single extension cycle. In one aspect, the extension cycle is stopped short at a predetermined point at which, on average, only 0, 1, 2, or 3 nucleotides have been incorporated into the primer, rather than permitting the reaction to run to near or full completion in each cycle.
- Short cycle methods according to the invention increase the resolution of individual nucleotides incorporated into the primer, but can decrease the yield of target templates successfully incorporating a nucleotide in a single extension cycle. In traditional full cycle sequencing, nucleotides may be allowed to react in the presence of a polymerizing agent until at least one becomes incorporated into at least 99% of the complementary strands. This would produce a yield of (0.99)n×100% for a complementary strand extended by n nucleotides. Obtaining incorporation in 99% of the complementary strands, however, requires a period of several half-lives of the incorporation reaction, where one half-life is the time taken for at least one incorporation to occur in 50% of the complementary strands. Typically, the more strands that complete an incorporation during each cycle, the more n-mers obtained after n cycles.
- According to the invention, short cycle methods rely on a period of only a limited number of half-lives of exposure to nucleotides, thus resulting in fewer target templates having incorporated a nucleotide in the short extension cycle. However, the short sequencing cycles provided by methods of the invention allow asynchronous analysis of polynucleotides. Thus, if an incorporation reactions fails to occur on a particular target polynucleotide, it can be completed in a later cycle without producing erroneous information, or interfering with data from other target molecules being analyzed in parallel. As demonstrated in
FIG. 1 , a cytosine (“C”) incorporates into the extension product of one copy of a target polynucleotide, but fails to incorporate into the other copy. During subsequent cycles of incorporation, however, a C can be incorporated, without adversely affection sequencing information. Thus, in asynchronous incorporation, an incorporation that failed to occur on a particular target in one-cycle can “catch up” in later cycles, permitting the use of shorter, even if more numerous, cycles. - Because short cycle methods according the invention permit the detection of, for example, one, two or three individual nucleotides incorporated into a primed template, the invention overcomes the difficulty posed by homopolymer regions of a template sequence. While detection techniques may be able to quantify signal intensity from a smaller number of incorporated nucleotides of the same base-type, for example two or three incorporated nucleotides, longer runs of identical bases may not permit quantification due to increasing signal intensity. That is, it may become difficult to distinguish n bases from n+1 bases, where the fractional increase in signal intensity from the (n+1)′h base is small relative to the signal intensity from the already-incorporated n bases.
- In embodiments using short-cycles, it is possible to limit the number of nucleotides that become incorporated in a given cycle. For example, it can be determined by simulation that using a cycle period of about 0.8 half-lives can result in two or less incorporations in nine out of ten homopolymer complementary strands. (See Example 2b). In another simulation, a 0.8 half-life period was shown to allow no more than two incorporations in about 96.0% of 200 homopolymer complementary strands. As detection means can more readily quantify signal intensity from the smaller number of incorporated nucleotides rather than from larger numbers, the use of short-cycles addresses this issue. For example, imaging systems known in the art can reliably distinguish the difference in signal intensity between one versus two fluorescent labeling moieties on consecutively-incorporated nucleotides. Other imaging systems can reliably distinguish the difference in signal intensity between two versus three fluorescent labeling moieties on consecutively-incorporated nucleotides.
- In a further embodiment of the invention, an extension cycle comprising a labeled nucleotide is followed by an extension cycle using an unlabeled nucleotide of the same type so that the position in each of the target template in which a labeled nucleotide failed to incorporated becomes occupied by an unlabeled nucleotide. Methods in accordance with this embodiment provide for continued participation of specific template nucleic acids in which no incorporation of the labeled nucleotide occurred and reduced probability of missing nucleotides in the resulting compiled sequence.
- Further methods of the invention provide for identifying a placeholder in a nucleic acid sequence in the event that an accurate determination of a nucleotide at a particular position is not possible. A placeholder is simply a position of unknown identity. Such a placeholder may be represented in a nucleic acid sequence with, for example, an “X,” a traditional symbol for an unspecified nucleotide. Slotting a placeholder in a nucleic acid sequence avoids frameshift-type errors in sequence determination.
- Additional aspects of the invention are described in the following sections and illustrated by the Examples.
- The invention is useful in sequencing any form of polynucleotides, including double-stranded DNA, single-stranded DNA, single-stranded DNA hairpins, DNA/RNA hybrids, RNAs with a recognition site for binding of the polymerizing agent, and RNA hairpins. Further, target polynucleotides may be a specific portion of a genome of a cell, such as an intron, regulatory region, allele, variant or mutation; the whole genome; or any portion therebetween. In other embodiments, the target polynucleotides may be mRNA, tRNA, rRNA, ribozymes, antisense RNA or RNAi. The target polynucleotide may be of any length, such as at least 10 bases, at least 25 bases, at least 50 bases, at least 100 bases, at least 500 bases, at least 1000 bases, or at least 2500 bases. The invention is particularly useful in high throughput sequencing of single molecule polynucleotides in which a plurality of target polynucleotides are attached to a solid support in a spatial arrangement such that each polynucleotides is individually optically resolvable. According to the invention, each detected incorporated label represents a single polynucleotide
- Nucleotides useful in the invention include both naturally-occurring and modified or non-naturally occurring nucleotides, and include nucleotide analogues. A nucleotide according to the invention may be, for example, a ribonucleotide, a deoxyribonucleotide, a modified ribonucleotide, a modified deoxyribonucleotide, a peptide nucleotide, a modified peptide nucleotide or a modified phosphate-sugar backbone nucleotide. Many aspects of nucleotides useful in the methods of the invention are subject to manipulation provide and suitable mechanisms for controlling the reaction. In particular, the species or type of nucleotide (i.e., natural or synthetic dATP, dCTP, dTTP, dGTP or dUTP; a natural or non-natural nucleotide) will affect the rate or efficiency of the reaction and therefore require consideration in preselecting parameters to produce the desire results.
- In addition, certain modifications to the nucleotides, including attaching a label, will affect the reaction. The size, polarity, hydrophobicity, hydrophilicity, charge, and other chemical attributes should be considered in determining parameters that will produce the desired results in the reaction. Labeled nucleotides of the invention include any nucleotide that has been modified to include a label which is directly or indirectly detectable. Such labels include optically-detectable labels such fluorescent labels, including fluorescein, rhodamine, phosphor, polymethadine dye, fluorescent phosphoramidite, texas red, green fluorescent protein, acridine, cyanine,
cyanine 5 dye,cyanine 3 dye, 5-(2′-aminoethyl)-aminonaphthalene-1-sulfonic acid (EDANS), BODIPY, ALEXA, or a derivative or modification of any of the foregoing. In one embodiment of the invention, fluorescence resonance energy transfer (FRET) technology is employed to produce a detectable, but quenchable, label. FRET may be used in the invention by, for example, modifying the primer to include a FRET donor moiety and using nucleotides labeled with a FRET acceptor moiety. - The fluorescently labeled nucleotides can be obtained commercially (e.g., from NEN DuPont, Amersham, and BDL). Alternatively, fluorescently labeled nucleotides can also be produced by various techniques, such as those described in Kambara et al., Bio/Techol. (1988) 6:816-821; Smith et al., Nucl. Acid Res. (1985) 13: 2399-2412, and Smith et al., Nature (1986) 321: 674-79.
- The fluorescent dye is preferably linked to the deoxyribose by a linker arm which is easily cleaved by chemical or enzymatic means. The length of the linker between the dye and the nucleotide can impact the incorporation rate and efficiency (see Zhu et al., Cytometry (1997) 28, 206). There are numerous linkers and methods for attaching labels to nucleotides, as shown in Oligonucleotides and Analogues: A Practical Approach (1991) (IRL Press, Oxford); Zuckerman et al., Polynucleotides Research (1987) 15: 5305-21; Sharma et al., Polynucleotides Research, (1991) 19: 3019; Giusti et al., PCR Methods and Applications (1993) 2: 223-227; Fung et al., U.S. Pat. No. 4,757,141; Stabinsky, U.S. Pat. No. 4,739,044; Agrawal et al., Tetrahedron Letters, (1990) 31: 1543-46; Sproat et al., Polynucleotides Research (1987) 15: 4837; and Nelson et al., Polynucleotides Research, (1989) 17: 7187-94.
- While the invention is exemplified herein with fluorescent labels, the invention is not so limited and can be practiced using nucleotides labeled with any form of detectable label, including radioactive labels, chemoluminescent labels, luminescent labels, phosphorescent labels, fluorescence polarization labels, and charge labels.
- Any parameter that permits the regulation of the number of labeled nucleotides added to the primer, or the rate at which the nucleotides are incorporated and detected can be controlled or exploited in the practice of the invention. Such parameters include, for example, the presence or absence of a label on a nucleotide, the type of label and manner of label attachment; the linker identity and length used to attach the label; the type of nucleotide (including, for example, whether such nucleotide is a dATP, dCTP, dTTP, dGTP or dUTP; a natural or non-natural nucleotide, a nucleotide analogue, or a modified nucleotide); the local sequence immediately 3′ to the addition position; whether the base is the first, second, third, etc. base added; the type of polymerase used; the particular batch characteristics of the polymerase; the processivity of the polymerase; the incorporation rate of the polymerase, and use of polymerase cofactors.
- In addition, a variety of the conditions of the reaction provide useful mechanisms for controlling either the number of nucleotides incorporated in a single extension reaction or the rates of nucleotide incorporation and detection. Such conditions include the “half-life” of the extension cycle (where one half-life is the time taken for at least one incorporation to occur in 50% of the complementary strands); the number of wash cycles (i.e., the number of times a nucleotide is introduced to the reaction then washed out); the number of target nucleic acids in the reaction; and the temperature of the reaction and the reagents used in the reaction.
- Half-Lives and Wash Cycles
- Based on the methods disclosed herein, those of skill in the art will be able to determine the period of half-lives required to limit the number incorporations per cycle for a given number of target polynucleotides. (See Examples 2 and 3,
FIGS. 2 and 3 ). Statistical simulations can also provide the number of repeated cycles needed to obtain a given number of incorporations, for example, to sequence a 25 base pair sequence. (See Examples 2 and 3,FIGS. 2 and 3 ). Referring to the simulations above, for example, it can be determined that 60 cycles, each 0.8 half-lives long, would be required for at least 25 incorporations in each of ten complementary strands (Example 2b,FIG. 2 b). With 200 complementary strands, 60 cycles each 0.8 half-lives long produce at least 20 incorporations in each strand (Example 3,FIG. 3 ). Following the methodologies outlined herein, such as the simulated working examples detailed below, those of skill in the art will be able to make similar determinations for other numbers of targets of varying lengths, and use appropriate cycle periods and numbers of cycles to analyze homopolymer without using blocking moieties or reversible chain termination. - The cycle period may also be chosen to permit a certain chance of incorporation of a given number of nucleotides in a complementary strand, and the cycle may be repeated a number of times to analyze the sequence of various numbers of target polynucleotides of varying length.
- In some embodiments, nucleotide half-lives for the incorporation reaction are affected by the fact that polymerizing agent may incorporate labeled nucleotides less readily than unlabeled nucleotides.
FIG. 4 illustrates the statistics of incorporation for a certain embodiment using a Klenow exo-minus polymerizing agent and Cy3- or Cy5-labeled nucleotides. The results show that polymerase may incorporate subsequent labeled nucleotides less readily than a prior labeled nucleotide. The graph ofFIG. 4 indicates, for example, that it may take five to ten times longer, resulting in a “stalling” of the incorporation reaction. In other embodiments, the stalling may vary with the use of other labeled nucleotides, other polymerizing agents and various reaction conditions. - Polymerase stalling is a useful mechanism for controlling incorporation rates in single molecule reactions. As is shown in the Examples below, polymerase stalling is useful to limit incorporation of nucleotides into any given strand in a fairly precise manner. According to the invention, polymerase stalling is useful to limit incorporation to 1 nucleotide per strand per cycle, on average. Given a priori knowledge of the statistics of incorporation, single molecule reactions are controlled to provide a statistical likelihood that 1, sometimes 2, but rarely 3 nucleotides are incorporated in a strand in any given cycle.
- For example, the rate at which polymerase incorporates labeled nucleotides into a complementary strand may be slowed by a factor of about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, or about 15 times compared to that observed with unlabeled nucleotides or compared to that observed for a prior incorporated labeled nucleotide.
- Moreover, this inhibition or delaying and longer half-lives can be taken into account when determining appropriate cycle periods and numbers of cycles to analyze homopolymer targets of a given length.
FIGS. 3 and 4 , for example, illustrate the results of simulations in which various factors affecting incorporation rates are taken into account. The graph ofFIG. 4 , for example, shows the number of cycles needed with cycle periods of various half-lives, taking into account stalling factors of two (squares), five (triangles), and 10 (crosses), in order to obtain 25 incorporations in over 80% of target strands, with at least a 97% chance of incorporating two or fewer nucleotides per cycle (or a smaller than 3% chance of incorporating three or more nucleotides per cycle). As the graph shows, stalling allows longer half-lives, which, in turn, permits the use of fewer cycles to obtain a “full” sequence with a defined error rate. AsFIG. 5 illustrates, if the use of labeled nucleotides slows down the polymerizing agent by a factor of 5, a cycle period of 2.4 half-lives produces over 80% 25-mers in 30 cycles. Based on the teachings of the invention, one of ordinary skill in the art can determine the cycle period required to limit the number incorporations per cycle for a given number of target polynucleotides of a given length. - Applying methods disclosed herein, the cycle period may be selected to permit about a 70%, about a 75%, about an 80%, about an 85%, about a 90%, about a 95%, about a 96%, about a 97%, about a 98%, and about a 99% chance of incorporation of two or less nucleotides into the complementary strand. Other cycle periods that may be used in embodiments of the invention include, for example, no more than about 5 half-lives, no more than about 4 half-lives, no more than about 3 half-lives, no more than about 2 half-lives, no more than about 1 half-lives, no more than about 0.9 half-lives, no more than about 0.8 half-lives, no more than about 0.7 half-lives, no more than about 0.6 half-lives, no more than about 0.5 half-lives, no more than about 0.4 half-lives, no more than about 0.3 half-lives, and no more than about 0.2 half-lives of the incorporation reactions.
- In addition to the Examples provided below, various cycle periods and number of times the cycles are repeated may be used with various numbers of targets in certain embodiments of the invention. These include, for example, using about 200 target polynucleotides, a period of no more than about 0.6 half-lives and repeating at least about 50 times; using about 200 target polynucleotides, a period of no more than about 0.6 half-lives and repeating at least about 60 times; using about 200 target polynucleotides, a period of no more than about 0.6 half-lives and repeating at least about 70 times; using about 200 target polynucleotides, a period of no more than about 0.8 half-lives and repeating at least about 50 times; using about 200 target polynucleotides, a period of no more than about 0.8 half-lives and repeating at least about 60 times; using about 200 target polynucleotides, a period of no more than about 0.8 half-lives and repeating at least about 70 times; using about 200 target polynucleotides, a period of no more than about 1 half-life and repeating at least about 50 times; using about 200 target polynucleotides, a period of no more than about 1 half-life and repeating at least about 60 times; and using about 200 target polynucleotides, a period of no more than about 1 half-life and repeating at least about 70 times. In any of these embodiments, signal from incorporated nucleotides may be reduced after each or a number of cycles.
- The number of times the cycles need to be repeated is also determined based on methods described herein. In general, the number of cycles increases with the length of the sequence to be analyzed and the duration of the half life of nucleotide exposure decreases as the length of sequence to be analyzed becomes longer. Also in general, half lives of nucleotide exposure increase and cycle numbers decrease with greater inhibitory or delaying effects on nucleotide incorporation
- Taking into account various stalling factors, examples of cycle periods and number repeat cycles that may be used in certain embodiments further include a cycle period of no more than about 0.5 half-lives with a stalling factor of about 2, repeated at least about 90 times; a cycle period of no more than about 0.75 half-lives, with a stalling factor of about 2, repeated at least about 75 times; a cycle period of no more than about 1 half-lives, with a stalling factor of about 2, repeated at least about 50 times; a cycle period of no more than about 1.5 half-lives with a stalling factor of about 2 or about 5, repeated at least about 45 times; a cycle period of no more than about 1.75 half-lives, with a stalling factor of about 5, repeated at least about 35 times; a cycle period of no more than about 2 half-lives, with a stalling factor of about 5 or about 10, repeated at least about 35 times; a cycle period of no more than about 2.25 half-lives, with a stalling factor of about 5 or about 10, repeated at least about 30 or at least about 35 times, and a cycle period of about 2.4 half-lives, with a stalling factor of about 5, repeated at least about 30 times.
- Polymerases and Polymerase Cofactors
- Polymerizing agents useful in the invention include DNA polymerases (such as Taq polymerase, T7 mutant DNA polymerase, Klenow and Sequenase, 9° N or a variant thereof), RNA polymerases, thermostable polymerases, thermodegradable polymerases, and reverse transcriptases. See e.g., Doublie et al., Nature (1998) 391:251-58; Ollis et al. Nature (1985) 313: 762-66; Beese et al., Science (1993) 260: 352-55; Korolev et al., Proc. Natl. Acad. Sci. USA (1995) 92: 9264-68; Keifer et al., Structure (1997) 5:95-108; and Kim et al., Nature (1995) 376:612-16.
- Cofactors of the invention function to inhibit the polymerizing agent, thereby slowing or stopping synthesis activity, permitting the detection of an incorporated labeled nucleotide. Cofactors of the invention include any chemical agent or reaction condition that results in the inhibition of the polymerizing agent. Such inhibition may be in whole or in part and may be permanent, temporary or reversible. For example, a cofactor may be a label, an antibody, an aptamer, an organic or inorganic small molecule, or a polyanion, or it may comprise a chemical modification to a nucleotide (i.e., a nucleotide analogue may comprise a cofactor). A cofactor can be in solution, or it may be attached, either directly or through a linker to a nucleotide, primer, template or polymerase.
- Examples of useful cofactor agents include, among others, light sensitive groups such as 6-nitoveratryloxycarbonyl (NVOC), 2-nitobenzyloxycarbonyl (NBOC), α,α-dimethyl-dimethoxybenzyloxycarbonyl (DDZ), 5-bromo-7-nitroindolinyl, o-hyrdoxy-2-methyl cinnamoyl, 2-oxymethylene anthraquinone, and t-butyl oxycarbonyl (TBOC). Oligonucleotide Synthesis: A Practical Approach (IRL Press, Oxford). Useful polyanions are described in U.S. Pat. No. 6,667,165 (the disclosure of which is incorporated by reference herein); and useful aptamers are described in U.S. Pat. Nos. 6,020,130 and 6,183,967 (the disclosures of which are incorporated by reference herein). See U.S. Pat. No. 5,338,671 for useful antibodies. Nucleotides possessing various labels and cofactors can be readily synthesized. Labeling moieties are attached at appropriate sites on the nucleotide using chemistry and conditions as described in Gait (1984).
- Further, the cofactor may also be the detectable label. Labels useful as combined labels/cofactors include larger or bulky dyes. For example, the detectable label may comprise a dye having a bulky chemical structure that, once the nucleotide is incorporated into the extending primer, causes a steric hindrance of the polymerizing agent, blocking the polymerizing agent from any further synthesis. Examples of labels that may be useful for this purpose are described in the Example, as well as in Zhu et al., Polynucleotides Res. (1994) 22: 3418-22. For example, fluorophore labels that may be used to stall the polymerase include Cy3, Cy5, Cy7, ALEXA647, ALEXA 488, BODIPY 576/589, BODIPY 650/665, BODIPY TR, Nile Blue, Sulfo-IRD700, NN382, R6G, Rho123, tetramethylrhodamine and Rhodamine X. In one embodiment, the labels are as bulky as Cy5, with molecular weights at least about 1.5 kDa. In another embodiment, the labels are bulkier than Cy5, having molecular weights of at least about 1.6 kDa, at least about 1.7 kDa, at least about 1.8 kDa, at least about 1.9 kDa, at least about 2.0 kDa at least about 2.5 kDa, or at least about 3.0 kDa.
- Further examples of such larger dyes include the following, with corresponding formula weights (in g/mol) in parentheses: Cy5 (534.6); Pyrene (535.6); 6-Carboxyfluorescein (FAM) (537.5); 6-Carboxyfluorescein-DMT (FAM-X (537.5); 5(6) Carboxyfluorescein (FAM) (537.5); 5-Fluorescein (FITC) (537.6); Cy3B (543.0); WellRED D4-PA (544.8); BODIPY 630/650 (545.5); 3′ 6-Carboxyfluorescein (FAM) (569.5); Cy3.5 (576.7); Cascade Blue (580.0); ALEXA Fluor 430 (586.8); Lucifer Yellow (605.5); ALEXA Fluor 532 (608.8); WellRED D2-PA (611.0); Cy5.5 (634.8); DY-630 (634.8); DY-555 (636.2); WellRED D3-PA (645.0); Rhodamine Red-X (654.0); DY-730 (660.9); DY-782 (660.9); DY-550 (667.8); DY-610 (667.8); DY-700 (668.9); 6-Tetrachlorofluorescein (TET) (675.2) ALEXA Fluor 568 (676.8); DY-650 (686.9); 5(6)-Carboxyeosin (689.0); Texas Red-X (702.0); ALEXA Fluor 594 (704.9); DY-675 (706.9); DY-750 (713.0); DY-681 (736.9); Hexachlorofluorescein (HEX) (744.1); DY-633 (751.9); LightCycler Red 705 (753.0); LightCycler Red 640 (758.0); DY-636 (760.9); DY-701 (770.9); FAR-Fuchsia (5′-Amidite) (776.0); FAR-Fuchsia (SE) (776.0); DY-676 (808.0); Erythrosin (814); FAR-Blue (5′-Amidite) (824.0); FAR-Blue (SE) (824.0); Oyster 556 (850.0); Oyster 656 (900.0); FAR-Green Two (SE) (960.0); ALEXA Fluor 546 (964.4); FAR-Green One (SE), (976.0); ALEXA Fluor 660 (985.0); Oyster 645 (1000.0); ALEXA Fluor 680 (1035.0); ALEXA Fluor 633 (1085.0); ALEXA Fluor 555 (1135.0); ALEXA Fluor 647 (1185.0); ALEXA Fluor 750 (1185.0); ALEXA Fluor 700 (1285.0). These reagents are commercially available from SYNTHEGEN, LLC (Houston, Tex.).
- There is extensive guidance in the literature for derivatizing fluorophore and quencher molecules for covalent attachment via common reactive groups that can be added to a nucleotide (see Haugland, Handbook of Fluorescent Probes and Research Chemicals (1992). There are also many linking moieties and methods for attaching fluorophore moieties to nucleotides, as described in Oligonucleotides and Analogues, supra; Guisti et al., supra; Agrawal et al, Tetrahedron Letters (1990) 31: 1543-46; and Sproat et al., Polynucleotide Research (1987) 15: 4837.
- In one embodiment, the method further comprises inactivating the cofactor, thereby reversing its effect on the polymerizing agent. Modes of inactivation depend on the cofactor. For example, where the cofactor is attached to the nucleotide, inactivation can typically be achieved by chemical, enzymatic, photochemical or radiation cleavage of the cofactor from the nucleotide. Cleavage of the cofactor can be achieved if a detachable connection between the nucleotide and the cofactor is used. For example, the use of disulfide bonds enables one to disconnect the dye by applying a reducing agent like dithiothreitol (DTT). In a further alternative, where the cofactor is a fluorescent label, it is possible to neutralize the label by bleaching it with radiation.
- In the event that temperature-sensitive cofactors are utilized, inactivation may comprise adjusting the reaction temperature. For example, an antibody that binds to thermostable polymerase at lower temperatures and blocks activity, but is denatured at higher temperatures, thus rendering the polymerase active; or single-stranded aptamers that bind to thermophilic polymerase at lower temperatures but are released at higher temperatures, may be inactivated by increasing the reaction temperature such the cofactor is released but polymerase activity is permitted.
- In one embodiment, transient inhibition of the polymerase and the time of exposure to the labeled nucleotide are coordinated such that it is statistically likely that at least one of the labeled nucleotide is incorporated in the primer, but statistically unlikely that more than two of the labeled nucleotide are incorporated. In another embodiment, the reaction is controlled by inhibiting the polymerase activity such that it is statistically unlikely that more than, for example, one or two nucleotides are incorporated into the same primer strand in the cycle.
- Temperature and Reagents
- Other reaction conditions that are useful in the methods of the invention include reaction temperature and reagents. For example, a temperature above or below the temperature required for optimal activity of the polymerizing agent, such as a temperature of about 20-70°, would be expected to result in a modulation of the polymerization rate, C. This form of inhibition is typically reversible with correction of the reaction temperature, provided that the delta in temperature was insufficient to cause a permanent damage to the polymerase.
- In another embodiment, buffer reagents useful in the methods of the invention include a detergent or surfactant, such as Triton®-
X 100, or salt and/or ion concentrations that facilitate or inhibit nucleotide incorporation. - The predetermined point at which a short cycle is stopped is defined, for example, by the occurrence of an event (such as the incorporation of a nucleotide comprising a blocking moiety that prevents further extension of the primer), the lapse of a certain amount of time (such as a specific number of half-lives), or the achievement of a statistically-significant datapoint (such as a period at which a statistically significant probability of two or less nucleotides have been incorporated). In one embodiment, the predetermined period of time is coordinated with an amount of polymerization inhibition such that, on average, a certain number of labeled nucleotides are added to the primer. In another embodiment, the number of incorporated labeled nucleotides is, on average, 0, 1 or 2, but almost never more than 3. The time period of exposure is defined in terms of statistical significance. For example, the time period may be that which is statistically insufficient for incorporation of more nucleotides than are resolvable by a detection system used to detect incorporation of the nucleotide into the primer. In another example, the time period that is statistically insufficient for incorporation of a greater number of nucleotides that are individually optically resolvable during a predetermined detection period (i.e., a period of time during which the incorporated nucleotides are detected).
- The reaction may be stopped by washing or flushing out the nucleotides that remain unincorporated and/or washing or flushing out polymerization agent. Further, many aspects of the repeated cycles may be automated, for example, using microfluidics for washing nucleotides to sites of anchored target polynucleotides, and washing out unincorporated nucleotides to halt each cycle.
- The following exemplifications of the invention are useful in understanding certain aspects of the invention but are not intended to limit the scope of the invention in any way.
- Primers are synthesized from nucleoside triphosphates by known automated oligonucleotide synthetic techniques, e.g., via standard phosphoramidite technology utilizing a nucleic acid synthesizer, such as the ABI3700 (Applied Biosystems, Foster City, Calif.). The oligonucleotides are prepared as duplexes with a complementary strand, however, only the 5′ terminus of the oligonucleotide proper (and not its complement) is biotinylated.
- Ligation of Oligonucleotides and Target Polynucleotides
- Double stranded target nucleic acids are blunt-end ligated to the oligonucleotides in solution using, for example, T4 ligase. The single strand having a 5′ biotinylated terminus of the oligonucleotide duplex permits the blunt-end ligation on only on end of the duplex. In a preferred embodiment, the solution-phase reaction is performed in the presence of an excess amount of oligonucleotide to prohibit the formation of concantamers and circular ligation products of the target nucleic acids. Upon ligation, a plurality of chimeric polynucleotide duplexes result. Chimeric polynucleotides are separated from unbound oligonucleotides based upon size and reduced to single strands by subjecting them to a temperature that destabilizes the hydrogen bonds.
- Preparation of Solid Support
- A solid support comprising reaction chambers having a fused silica surface is sonicated in 2% MICRO-90 soap (Cole-Parmer, Vernon Hills, Ill.) for 20 minutes and then cleaned by immersion in boiling RCA solution (6:4:1 high-purity H2O/30% NH4OH/30% H2O2) for 1 hour. It is then immersed alternately in polyallylamine (positively charged) and polyacrylic acid (negatively charged; both from Aldrich) at 2 mg/ml and
pH 8 for 10 minutes each and washed intensively with distilled water in between. The slides are incubated with 5 mM biotin-amine reagent (Biotin-EZ-Link, Pierce) for 10 minutes in the presence of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC, Sigma) in MES buffer, followed by incubation with Streptavidin Plus (Prozyme, San Leandro, Calif.) at 0.1 mg/ml for 15 minutes in Tris buffer. The biotinylated single-stranded chimeric polynucleotides are deposited via ink jet printing onto the streptavidin-coated chamber surface at 10 pM for 10 minutes in Tris buffer that contain 100 mM MgCl2. - Equipment
- The experiments are performed on an upright microscope (BH-2, Olympus, Melville, N.Y.) equipped with total internal reflection (TIR) illumination, such as the BH-2 microscope from Olympus (Melville, N.Y.). Two laser beams, 635 (Coherent, Santa Clara, Calif.) and 532 nm (Brimrose, Baltimore), with nominal powers of 8 and 10 mW, respectively, are circularly polarized by quarter-wave plates and undergo TIR in a dove prism (Edmund Scientific, Barrington, N.J.). The prism is optically coupled to the fused silica bottom (Esco, Oak Ridge, N.J.) of the reaction chambers so that evanescent waves illuminated up to 150 nm above the surface of the fused silica. An objective (DPlanApo, 100 UV 1.3oil, Olympus) collects the fluorescence signal through the top plastic cover of the chamber, which is deflected by the objective to ≈40μm from the silica surface. An image splitter (Optical Insights, Santa Fe, N.M.) directs the light through two bandpass filters (630dcxr, HQ585/80, HQ690/60; Chroma Technology, Brattleboro, Vt.) to an intensified charge-coupled device (I-PentaMAX; Roper Scientific, Trenton, N.J.), which records adjacent images of a 120-×60-μm section of the surface in two colors.
- Experimental Protocols
- FRET-Based Method Using Nucleotide-Based Donor Fluorophore
- In a first experiment, universal primer is hybridized to a primer attachment site present in support-bound chimeric polynucleotides. Next, a series of incorporation reactions are conducted in which a first nucleotide comprising a cyanine-3 donor fluorophore is incorporated into the primer as the first extended nucleotide. If all the chimeric sequences are the same, then a minimum of one labeled nucleotide must be added as the initial FRET donor because the template nucleotide immediately 3′ of the primer is the same on all chimeric polynucleotides. If different chimeric polynucleotides are used (i.e., the polynucleotide portion added to the bound oligonucleotides is different at least one location), then all four labeled dNTPs initially are cycled. The result is the addition of at least one donor fluorophore to each chimeric strand.
- The number of initial incorporations containing the donor fluorophore is limited by either limiting the reaction time (i.e., the time of exposure to donor-labeled nucleotides), by polymerase stalling, or both in combination. The inventors have shown that base-addition reactions are regulated by controlling reaction conditions. For example, incorporations can be limited to 1 or 2 at a time by causing polymerase to stall after the addition of a first base. One way in which this is accomplished is by attaching a dye to the first added base that either chemically or sterically interferes with the efficiency of incorporation of a second base. A computer model was constructed using Visual Basic (v. 6.0, Microsoft Corp.) that replicates the stochastic addition of bases in template-dependent nucleic acid synthesis. The model utilizes several variables that are thought to be the most significant factors affecting the rate of base addition. The number of half-lives until dNTPs are flushed is a measure of the amount of time that a template-dependent system is exposed to dNTPs in solution. The more rapidly dNTPs are removed from the template, the lower will be the incorporation rate. The number of wash cycles does not affect incorporation in any given cycle, but affects the number bases ultimately added to the extending primer. The number of strands to be analyzed is a variable of significance when there is not an excess of dNTPs in the reaction. Finally, the inhibition rate is an approximation of the extent of base addition inhibition, usually due to polymerase stalling. The homopolymer count within any strand can be ignored for purposes of this application.
FIG. 2 is a screenshot showing the inputs used in the model. - The model demonstrates that, by controlling reaction conditions, one can precisely control the number of bases that are added to an extending primer in any given cycle of incorporation. For example, as shown in
FIG. 7 , at a constant rate of inhibition of second base incorporation (i.e., the inhibitory effect of incorporation of a second base given the presence of a first base), the amount of time that dNTPs are exposed to template in the presence of polymerase determines the number of bases that are statistically likely to be incorporated in any given cycle (a cycle being defined as one round of exposure of template to dNTPs and washing of unbound dNTP from the reaction mixture). As shown inFIG. 7 a, when time of exposure to dNTPs is limited, the statistical likelihood of incorporation of more than two bases is essentially zero, and the likelihood of incorporation of two bases in a row in the same cycle is very low. If the time of exposure is increased, the likelihood of incorporation of multiple bases in any given cycle is much higher. Thus, the model reflects biological reality. At a constant rate of polymerase inhibition (assuming that complete stalling is avoided), the time of exposure of a template to dNTPs for incorporation is a significant factor in determining the number of bases that will be incorporated in succession in any cycle. Similarly, if time of exposure is held constant, the amount of polymerase stalling will have a predominant effect on the number of successive bases that are incorporated in any given cycle (See,FIG. 7 b). Thus, it is possible at any point in the sequencing process to add or renew donor fluorophore by simply limiting the statistical likelihood of incorporation of more than one base in a cycle in which the donor fluorophore is added. - Upon introduction of a donor fluorophore into the extending primer sequence, further nucleotides comprising acceptor fluorophores (here, cyanine-5) are added in a template-dependent manner. It is known that the Foster radius of Cy-3/Cy5 fluorophore pairs is about 5 inn (or about 15 nucleotides, on average). Thus, donor must be refreshed about every 15 bases. This is accomplished under the parameters outlined above. In general, each cycle preferably is regulated to allow incorporation of 1 or 2, but never 3 bases. So, refreshing the donor means simply the addition of all four possible nucleotides in a mixed-sequence population using the donor fluorophore instead of the acceptor fluorophore every approximately 15 bases (or cycles).
FIG. 2 shows schematically the process of FRET-based, template-dependent nucleotide addition as described in this example. - The methods described above are alternatively conducted with the FRET donor attached to the polymerase molecule. In that embodiment, donor follows the extending primer as new nucleotides bearing acceptor fluorophores are added. Thus, there typically is no requirement to refresh the donor. In another embodiment, the same methods are carried out using a nucleotide binding protein (e.g., DNA binding protein) as the carrier of a donor fluorophore. In that embodiment, the DNA binding protein is spaced at intervals (e.g., about 5 nm or less) to allow FRET. Thus, there are many alternatives for using FRET to conduct single molecule sequencing using the devices and methods taught in the application. However, it is not required that FRET be used as the detection method. Rather, because of the intensities of the FRET signal with respect to background, FRET is an alternative for use when background radiation is relatively high.
- Non-FRET Based Methods
- Methods for detecting single molecule incorporation without FRET are also conducted. In this embodiment, incorporated nucleotides are detected by virtue of their optical emissions after sample washing. Primers are hybridized to the primer attachment site of bound chimeric polynucleotides Reactions are conducted in a solution comprising Klenow fragment Exo-minus polymerase (New England Biolabs) at 10 nM (100 units/nil) and a labeled nucleotide triphosphate in EcoPol reaction buffer (New England Biolabs). Sequencing reactions takes place in a stepwise fashion. First, 0.2 μM dUTP-Cy3 and polymerase are introduced to support-bound chimeric polynucleotides, incubated for 6 to 15 minutes, and washed out. Images of the surface are then analyzed for primer-incorporated U-Cy5. Typically, eight exposures of 0.5 seconds each are taken in each field of view in order to compensate for possible intermittency (e.g., blinking) in fluorophore emission. Software is employed to analyze the locations and intensities of fluorescence objects in the intensified charge-coupled device pictures. Fluorescent images acquired in the WinView32 interface (Roper Scientific, Princeton, N.J.) are analyzed using ImagePro Plus software (Media Cybernetics, Silver Springs, Md.). Essentially, the software is programmed to perform spot-finding in a predefined image field using user-defined size and intensity filters. The program then assigns grid coordinates to each identified spot, and normalizes the intensity of spot fluorescence with respect to background across multiple image frames. From those data, specific incorporated nucleotides are identified. Generally, the type of image analysis software employed to analyze fluorescent images is immaterial as long as it is capable of being programmed to discriminate a desired signal over background. The programming of commercial software packages for specific image analysis tasks is known to those of ordinary skill in the art. If U-Cy5 is not incorporated, the substrate is washed, and the process is repeated with dGTP-Cy5, dATP-Cy5, and dCTP-Cy5 until incorporation is observed. The label attached to any incorporated nucleotide is neutralized, and the process is repeated. To reduce bleaching of the fluorescence dyes, an oxygen scavenging system can be used during all green illumination periods, with the exception of the bleaching of the primer tag.
- In order to determine a template sequence, the above protocol is performed sequentially in the presence of a single species of labeled dATP, dGTP, dCTP or dUTP. By so doing, a first sequence can be compiled that is based upon the sequential incorporation of the nucleotides into the extended primer. The first compiled sequence is representative of the complement of the template. As such, the sequence of the template can be easily determined by compiling a second sequence that is complementary to the first sequence. Because the sequence of the oligonucleotide is known, those nucleotides can be excluded from the second sequence to produce a resultant sequence that is representative of the target template.
-
FIG. 2 illustrates the advantage of short-cycle sequencing with respect to avoiding long homopolymer reads.FIG. 2 a illustrates a simulated analysis of 10 target polynucleotides using non-short-cycle sequencing (Example 2a), whereasFIG. 2 b illustrates a simulated analysis of the same number of target polynucleotides using short-cycle sequencing (Example 2b). - The simulations were performed as follows: an Excel spreadsheet was opened and “Customize . . . ” selected from the “Tools” menu of the Excel toolbar. The “Commands” tab was selected and, after scrolling down, “Macros” was clicked. The “smiley face” that appeared in the right panel was dragged to the toolbars on top of the spreadsheet. The “Customize” box was closed and the “smiley face” clicked once. From the list of subroutines that appeared, “ThisWorkbook.Main_Line.” was selected. The program was run by clicking again on the “smiley face.” A copy of the source code for the Excel simulation is provided below.
- Input values were then entered into the tabbed sheet called “In Out.” There were three input values:
- The first input value corresponded to the period of time allowed for incorporation reactions of provided nucleotides into the growing complementary strands of the polynucleotides to be analyzed. This period was conveniently measured in half-lives of the incorporation reaction itself. Each cycle of incorporation was simulatedly stalled after a period of time, representing, for example, the time when unincorporated nucleotides would be flushed out or the incorporation reactions otherwise stalled.
- The second input value corresponds to the number of times each cycle of incorporation was repeated. That is, the number of times the steps of providing nucleotides, allowing incorporation reactions into the complementary strands in the presence of polymerizing agent, and then stopping the incorporations are repeated. The nucleotides were simulatedly provided as a wash of each of dATPs, dGTPs, dTTPs, and dCTPs. The program then recorded which nucleotides were incorporated, corresponding to a detection step of detecting incorporation.
- The third input value corresponds to number of strands of target polynucleotides to by analyzed in the simulation. The program allowed up to 1100 target polynucleotide molecules to be analyzed in a given simulation.
- After the program was started, as described above, the program first generated the inputted number of strands composed of random sequences. The program then simulated hybridization and polymerization of the correct base of each incorporation reaction, based on the generated sequence of the target polynucleotide templates. The program continued these simulated reactions for the allowed amount of simulated time, determined by the inputted number of half-lives. Statistics of the simulation were then computed and reported, including the longest strand, the shortest strand, and the average length of all strands, as well as the fraction of strands extended by at least 25 nucleotide incorporations, as discussed in more detail below.
- In the first part of this simulation, Example 2a, the input values used were a cycle period of 10 half-lives, 12 repeats of the cycle, and 10 target polynucleotide strands.
-
FIG. 2 a illustrates the results obtained. Homopolymers stretches which occurred in the same simulated complementary strand are highlighted in magenta wherever 2 nucleotides of the same base type were incorporated in a row, and in cyan wherever more than two nucleotides of the same base type were incorporated in a row. -
FIG. 2 a illustrates that the output values included the longest extended complementary strand obtained during the simulation (Longest extension in the ensemble of molecules); the shorted extended complementary strand obtained during the simulation (Shortest extension in the ensemble of molecules); and the average extension. These numbers represent the greatest number of incorporations into any of the 10 simulatedly growing complementary strands, the smallest number of incorporations for any of the 10, and the average number of incorporations for the 10.FIG. 2 a indicates that the values obtained for Example 2a were 37 incorporations in the longest extension, 25 in the shortest, and 30.00 as the average number of incorporations. - The output values also provided information on the number of incorporations that occurred in each of growing complementary strands during each cycle period of the simulation. For example,
FIG. 2 a indicates that for the input values of Example 2a, the percentage of growing stands extended by two or more nucleotides in a homopolymer stretch was 100.0%; and the percentage of growing strands extended by three or more nucleotides in a homopolymer stretch was 60.0%. That is, using a cycle period of 10 half-lives resulted in only 40% of the complementary strands being extended by two or less nucleotides in a homopolymer stretch per cycle of incorporation. - Further, output values also indicated the total number of incorporations for each of the growing strands for the total number of repeated cycles. This represents the length of the sequence of target polynucleotide analyzed.
FIG. 2 a illustrates that in Example 2a, 100.0% of the 10 target polynucleotides of the simulation were extended by at least 25 incorporated nucleotides. This illustrates that using a cycle period of 10 half-lives, and repeating thecycles 12 times, allowed analysis of a 25 base sequence of 10 target polynucleotides. - In the second part of this simulation, Example 2b, the input values used were a cycle period of 0.8 half-lives, 60 repeats of the cycle, and 10 target polynucleotide strands.
-
FIG. 2 b illustrates the results obtained. Homopolymers stretches which occurred in the same simulated complementary strand are highlighted in magenta wherever 2 nucleotides of the same base type were incorporated in a row, and in cyan wherever more than two nucleotides of the same base type were incorporated in a row. -
FIG. 2 b illustrates that the output values included the longest extended complementary strand obtained during the simulation (longest extension in the ensemble of molecules); the shortest extended complementary strand obtained during the simulation (shortest extension in the ensemble of molecules); and the average extension. These numbers represent the greatest number of incorporations into any of the 10 simulatedly growing complementary strands, the smallest number of incorporations for any of the 10, and the average number of incorporations for the 10.FIG. 2 b indicates that the values obtained for Example 2b were 37 incorporations in the longest extension, 26 in the shortest, and 32.00 as the average number of incorporations. - The output values also provided information on the number of incorporations that occurred in each of growing complementary strands during each cycle period of the simulation. For example,
FIG. 2 b indicates that for the input values of Example 2b, the percentage of growing stands extended by two or more nucleotides in a homopolymer stretch was 80.0%; and the percentage of growing strands extended by three or more nucleotides in a homopolymer stretch was 10.0%. That is, using a cycle period of 0.8 half-lives resulted in 90% of the complementary strands being extended by two or less nucleotides per cycle of incorporation. - Output values also indicated the total number of incorporations for each of the growing strands for the total number of repeated cycles. As in Example 2a, this represents the length of the sequence of target polynucleotide analyzed.
FIG. 2 b illustrates that in Example 2b, 100.0% of the 10 target polynucleotides of the simulation were again extended by at least 25 incorporated nucleotides. This illustrates that using a cycle period of 0.8 half-lives, and repeating thecycles 60 times, allowed analysis of a 25 base sequence of 10 target polynucleotides. - Comparing the two simulations, it will be appreciated by those in the art that the use of short-cycles of sequencing overcame issues of reading long repeats of homopolymer stretches in sequencing by synthesis, without using blocking moieties, as only a few nucleotides were incorporated per cycle. Comparing Examples 2a and 2b, the long cycles in 2a resulted in 40% of the extended complementary strands having two or less homopolymer nucleotide incorporations per cycle. Conversely, the short cycles in 11b resulted in 90% of the extended complementary strands having two or less homopolymer nucleotide incorporations per cycle, facilitating quantification. That is, as explained more thoroughly above, shorter reads can be quantitated to determine the number of nucleotides incorporated, for example, where the nucleotides are of the same
- Comparing Examples 2a and 2b also indicated that a greater number of repeated cycles were needed to analyze a given length of sequence when using shorter cycles. That is, the 10 half-lives cycle was repeated 12 times to result in 100.0% of the 10 complementary strands being extended by at least 25 nucleotides, whereas the 0.8 half-lives cycle was repeated 60 times to obtain this same result and thereby analyze the 25 nucleotides sequence.
- Nonetheless, many aspects of the repeated cycles may be automated, for example, using micro fluidics for washing nucleotides to sites of anchored target polynucleotides, and washing out unincorporated nucleotides to halt each cycle.
- As discussed herein, below is a copy of the source code for the simulation of short-cycle sequencing.
-
FIG. 2 illustrates yet another simulated analysis of a number of target polynucleotides using short-cycle sequencing. The simulation was run using the program described in Examples 2a and 2b but using a larger number of target polynucleotides. - That is, in this simulation, the input values used were a cycle period of 0.8 half-lives, 60 repeats of the cycle, and 200 target polynucleotide strands.
FIG. 2 illustrates the results obtained. Homopolymers stretches which occurred in the same simulated complementary strand are highlighted in magenta wherever nucleotides of the same base type were incorporated in a row, and in cyan wherever more than two nucleotides of the same base type were incorporated in a row. - The output values obtained were 48 incorporations in the longest extended complementary strand, 20 in the shortest, and 32.00 as the average number of incorporations for the 200 simulatedly extended complementary strands.
- Further, the percentage of growing stands extended by two or more nucleotides in a homopolymer stretch was 78.5%; and the percentage of growing strands extended by three or more nucleotides in a homopolymer stretch was 4.0%. That is, using a cycle period of 0.8 half-lives resulted in 96.0% of the complementary strands being extended by two or less nucleotides in a homopolymer stretch per cycle of incorporation. Moreover, 95.5% of the 200 target polynucleotides of the simulation were extended by at least 25 incorporated nucleotides, while 100% were extended by at least 20 nucleotides. This illustrated that using a cycle period of 0.8 half-lives, and repeating the
cycles 60 times, allows analysis of a 20 base sequence of 200 target polynucleotides. - This example demonstrates a method according to the invention in which a single nucleotide in a position in a nucleic acid sequence is identified. A template-bound primer is sequentially exposed first to a labeled nucleotide and then to an unlabeled nucleotide of the same type under conditions and in the presence of reagents that allow template-dependent primer extension. The template is analyzed in order to determine whether the first nucleotide is incorporated in the primer at the first position or not. If not, then the sequential exposure to labeled and unlabeled nucleotides is repeated using another type of nucleotide until one such nucleotide is determined to have incorporated at the first position. Once an incorporated nucleotide is determined, the identity of the nucleotide in the position in the nucleic acid sequence is identified as the complementary nucleotide.
- In this example, a series of reactions are performed as described above in Example 1. A nucleic acid primer is hybridized to a target nucleic acid at a primer binding site in the target. The primer comprises a donor fluorophore. The hybridized primer is exposed to a first nucleotide comprising an acceptor fluorophore comprising a blocking moiety that, when incorporated into the primer, prevents further polymerization of the primer. The presence or absence of fluorescent emission from each of the donor and the acceptor is determined. A nucleotide that has been incorporated into the primer via complementary base pairing with the target is identified by the presence of fluorescent emission from the acceptor, and a sequence placeholder is identified as the absence of fluorescent emission from the donor and the acceptor. A sequence of the target nucleic acid is complied based upon the sequence of the incorporated nucleotides and the placeholders.
Claims (8)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US13/008,468 US20110151449A1 (en) | 2003-11-12 | 2011-01-18 | Short cycle methods for sequencing polynucleotides |
Applications Claiming Priority (7)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US51986203P | 2003-11-12 | 2003-11-12 | |
US54627704P | 2004-02-19 | 2004-02-19 | |
US54761104P | 2004-02-24 | 2004-02-24 | |
US10/852,482 US7169560B2 (en) | 2003-11-12 | 2004-05-24 | Short cycle methods for sequencing polynucleotides |
US11/588,108 US7491498B2 (en) | 2003-11-12 | 2006-10-26 | Short cycle methods for sequencing polynucleotides |
US12/371,310 US7897345B2 (en) | 2003-11-12 | 2009-02-13 | Short cycle methods for sequencing polynucleotides |
US13/008,468 US20110151449A1 (en) | 2003-11-12 | 2011-01-18 | Short cycle methods for sequencing polynucleotides |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/371,310 Continuation US7897345B2 (en) | 2003-11-12 | 2009-02-13 | Short cycle methods for sequencing polynucleotides |
Publications (1)
Publication Number | Publication Date |
---|---|
US20110151449A1 true US20110151449A1 (en) | 2011-06-23 |
Family
ID=34595948
Family Applications (7)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US10/852,482 Expired - Fee Related US7169560B2 (en) | 2003-11-12 | 2004-05-24 | Short cycle methods for sequencing polynucleotides |
US11/588,108 Expired - Fee Related US7491498B2 (en) | 2003-11-12 | 2006-10-26 | Short cycle methods for sequencing polynucleotides |
US12/371,310 Expired - Fee Related US7897345B2 (en) | 2003-11-12 | 2009-02-13 | Short cycle methods for sequencing polynucleotides |
US13/008,468 Abandoned US20110151449A1 (en) | 2003-11-12 | 2011-01-18 | Short cycle methods for sequencing polynucleotides |
US13/008,182 Expired - Fee Related US9012144B2 (en) | 2003-11-12 | 2011-01-18 | Short cycle methods for sequencing polynucleotides |
US13/008,130 Abandoned US20110245086A1 (en) | 2003-11-12 | 2011-01-18 | Short cycle methods for sequencing polynucleotides |
US14/663,010 Expired - Fee Related US9657344B2 (en) | 2003-11-12 | 2015-03-19 | Short cycle methods for sequencing polynucleotides |
Family Applications Before (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US10/852,482 Expired - Fee Related US7169560B2 (en) | 2003-11-12 | 2004-05-24 | Short cycle methods for sequencing polynucleotides |
US11/588,108 Expired - Fee Related US7491498B2 (en) | 2003-11-12 | 2006-10-26 | Short cycle methods for sequencing polynucleotides |
US12/371,310 Expired - Fee Related US7897345B2 (en) | 2003-11-12 | 2009-02-13 | Short cycle methods for sequencing polynucleotides |
Family Applications After (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US13/008,182 Expired - Fee Related US9012144B2 (en) | 2003-11-12 | 2011-01-18 | Short cycle methods for sequencing polynucleotides |
US13/008,130 Abandoned US20110245086A1 (en) | 2003-11-12 | 2011-01-18 | Short cycle methods for sequencing polynucleotides |
US14/663,010 Expired - Fee Related US9657344B2 (en) | 2003-11-12 | 2015-03-19 | Short cycle methods for sequencing polynucleotides |
Country Status (4)
Country | Link |
---|---|
US (7) | US7169560B2 (en) |
EP (1) | EP1692312A4 (en) |
CA (1) | CA2545619A1 (en) |
WO (1) | WO2005047523A2 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20070099212A1 (en) * | 2005-07-28 | 2007-05-03 | Timothy Harris | Consecutive base single molecule sequencing |
US9012144B2 (en) | 2003-11-12 | 2015-04-21 | Fluidigm Corporation | Short cycle methods for sequencing polynucleotides |
Families Citing this family (480)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA2281205A1 (en) * | 1997-02-12 | 1998-08-13 | Eugene Y. Chan | Methods and products for analyzing polymers |
US6780591B2 (en) | 1998-05-01 | 2004-08-24 | Arizona Board Of Regents | Method of determining the nucleotide sequence of oligonucleotides and DNA molecules |
US7875440B2 (en) | 1998-05-01 | 2011-01-25 | Arizona Board Of Regents | Method of determining the nucleotide sequence of oligonucleotides and DNA molecules |
DE69930310T3 (en) | 1998-12-14 | 2009-12-17 | Pacific Biosciences of California, Inc. (n. d. Ges. d. Staates Delaware), Menlo Park | KIT AND METHOD FOR THE NUCLEIC ACID SEQUENCING OF INDIVIDUAL MOLECULES BY POLYMERASE SYNTHESIS |
WO2002044425A2 (en) | 2000-12-01 | 2002-06-06 | Visigen Biotechnologies, Inc. | Enzymatic nucleic acid synthesis: compositions and methods for altering monomer incorporation fidelity |
US20040161741A1 (en) | 2001-06-30 | 2004-08-19 | Elazar Rabani | Novel compositions and processes for analyte detection, quantification and amplification |
US9777312B2 (en) | 2001-06-30 | 2017-10-03 | Enzo Life Sciences, Inc. | Dual polarity analysis of nucleic acids |
US7668697B2 (en) * | 2006-02-06 | 2010-02-23 | Andrei Volkov | Method for analyzing dynamic detectable events at the single molecule level |
AU2004214891B2 (en) | 2003-02-26 | 2010-01-07 | Complete Genomics, Inc. | Random array DNA analysis by hybridization |
GB0307428D0 (en) * | 2003-03-31 | 2003-05-07 | Medical Res Council | Compartmentalised combinatorial chemistry |
US20060078893A1 (en) | 2004-10-12 | 2006-04-13 | Medical Research Council | Compartmentalised combinatorial chemistry by microfluidic control |
GB0307403D0 (en) | 2003-03-31 | 2003-05-07 | Medical Res Council | Selection by compartmentalised screening |
EP2248911A1 (en) * | 2004-02-19 | 2010-11-10 | Helicos Biosciences Corporation | Methods and kits for analyzing polynucleotide sequences |
US20100216153A1 (en) | 2004-02-27 | 2010-08-26 | Helicos Biosciences Corporation | Methods for detecting fetal nucleic acids and diagnosing fetal abnormalities |
US20050221339A1 (en) * | 2004-03-31 | 2005-10-06 | Medical Research Council Harvard University | Compartmentalised screening by microfluidic control |
US20050239085A1 (en) * | 2004-04-23 | 2005-10-27 | Buzby Philip R | Methods for nucleic acid sequence determination |
US20070117104A1 (en) * | 2005-11-22 | 2007-05-24 | Buzby Philip R | Nucleotide analogs |
US7833709B2 (en) | 2004-05-28 | 2010-11-16 | Wafergen, Inc. | Thermo-controllable chips for multiplex analyses |
WO2005123957A2 (en) * | 2004-06-10 | 2005-12-29 | Ge Healthcare Bio-Sciences Corp. | Method for nucleic acid analysis |
US8536661B1 (en) | 2004-06-25 | 2013-09-17 | University Of Hawaii | Biosensor chip sensor protection methods |
WO2006022370A1 (en) * | 2004-08-27 | 2006-03-02 | National Institute For Materials Science | Method of analyzing dna sequence using field-effect device, and base sequence analyzer |
US7968287B2 (en) | 2004-10-08 | 2011-06-28 | Medical Research Council Harvard University | In vitro evolution in microfluidic systems |
WO2006094149A2 (en) * | 2005-03-01 | 2006-09-08 | Exact Sciences Corporation | Methods and compositions for detecting adenoma |
SG162795A1 (en) | 2005-06-15 | 2010-07-29 | Callida Genomics Inc | Single molecule arrays for genetic and chemical analysis |
US7666593B2 (en) | 2005-08-26 | 2010-02-23 | Helicos Biosciences Corporation | Single molecule sequencing of captured nucleic acids |
US20090305248A1 (en) * | 2005-12-15 | 2009-12-10 | Lander Eric G | Methods for increasing accuracy of nucleic acid sequencing |
US20070154921A1 (en) * | 2005-12-16 | 2007-07-05 | Applera Corporation | Method and System for Phase-Locked Sequencing |
EP2363205A3 (en) | 2006-01-11 | 2014-06-04 | Raindance Technologies, Inc. | Microfluidic Devices And Methods Of Use In The Formation And Control Of Nanoreactors |
US20070196832A1 (en) * | 2006-02-22 | 2007-08-23 | Efcavitch J William | Methods for mutation detection |
SG10201405158QA (en) | 2006-02-24 | 2014-10-30 | Callida Genomics Inc | High throughput genome sequencing on dna arrays |
US20090075252A1 (en) * | 2006-04-14 | 2009-03-19 | Helicos Biosciences Corporation | Methods for increasing accuracy of nucleic acid sequencing |
US7702468B2 (en) | 2006-05-03 | 2010-04-20 | Population Diagnostics, Inc. | Evaluating genetic disorders |
US10522240B2 (en) | 2006-05-03 | 2019-12-31 | Population Bio, Inc. | Evaluating genetic disorders |
EP2530168B1 (en) | 2006-05-11 | 2015-09-16 | Raindance Technologies, Inc. | Microfluidic Devices |
US9562837B2 (en) | 2006-05-11 | 2017-02-07 | Raindance Technologies, Inc. | Systems for handling microfludic droplets |
US8137626B2 (en) * | 2006-05-19 | 2012-03-20 | California Institute Of Technology | Fluorescence detector, filter device and related methods |
EP4108780A1 (en) | 2006-06-14 | 2022-12-28 | Verinata Health, Inc. | Rare cell analysis using sample splitting and dna tags |
US20080050739A1 (en) | 2006-06-14 | 2008-02-28 | Roland Stoughton | Diagnosis of fetal abnormalities using polymorphisms including short tandem repeats |
EP2029779A4 (en) | 2006-06-14 | 2010-01-20 | Living Microsystems Inc | Use of highly parallel snp genotyping for fetal diagnosis |
EP2024512A4 (en) | 2006-06-14 | 2009-12-09 | Artemis Health Inc | Methods for the diagnosis of fetal abnormalities |
US8372584B2 (en) | 2006-06-14 | 2013-02-12 | The General Hospital Corporation | Rare cell analysis using sample splitting and DNA tags |
WO2008021123A1 (en) | 2006-08-07 | 2008-02-21 | President And Fellows Of Harvard College | Fluorocarbon emulsion stabilizing surfactants |
US8656949B2 (en) | 2006-08-15 | 2014-02-25 | University Of Maryland College Park | Microfluidic devices and methods of fabrication |
WO2008027558A2 (en) | 2006-08-31 | 2008-03-06 | Codon Devices, Inc. | Iterative nucleic acid assembly using activation of vector-encoded traits |
WO2008036614A1 (en) | 2006-09-18 | 2008-03-27 | California Institute Of Technology | Apparatus for detecting target molecules and related methods |
EP2071927A2 (en) | 2006-09-28 | 2009-06-24 | Illumina, Inc. | Compositions and methods for nucleotide sequencing |
US7910354B2 (en) | 2006-10-27 | 2011-03-22 | Complete Genomics, Inc. | Efficient arrays of amplified polynucleotides |
US20090111705A1 (en) | 2006-11-09 | 2009-04-30 | Complete Genomics, Inc. | Selection of dna adaptor orientation by hybrid capture |
US8349167B2 (en) | 2006-12-14 | 2013-01-08 | Life Technologies Corporation | Methods and apparatus for detecting molecular interactions using FET arrays |
CA2672315A1 (en) | 2006-12-14 | 2008-06-26 | Ion Torrent Systems Incorporated | Methods and apparatus for measuring analytes using large scale fet arrays |
US8262900B2 (en) * | 2006-12-14 | 2012-09-11 | Life Technologies Corporation | Methods and apparatus for measuring analytes using large scale FET arrays |
US11339430B2 (en) | 2007-07-10 | 2022-05-24 | Life Technologies Corporation | Methods and apparatus for measuring analytes using large scale FET arrays |
WO2008097559A2 (en) | 2007-02-06 | 2008-08-14 | Brandeis University | Manipulation of fluids and reactions in microfluidic systems |
WO2008130623A1 (en) | 2007-04-19 | 2008-10-30 | Brandeis University | Manipulation of fluids, fluid components and reactions in microfluidic systems |
US9163053B2 (en) * | 2007-05-18 | 2015-10-20 | Fluidigm Corporation | Nucleotide analogs |
US7678894B2 (en) * | 2007-05-18 | 2010-03-16 | Helicos Biosciences Corporation | Nucleotide analogs |
US8431367B2 (en) | 2007-09-14 | 2013-04-30 | Predictive Biosciences Corporation | Detection of nucleic acids and proteins |
US8222040B2 (en) * | 2007-08-28 | 2012-07-17 | Lightspeed Genomics, Inc. | Nucleic acid sequencing by selective excitation of microparticles |
US8759077B2 (en) * | 2007-08-28 | 2014-06-24 | Lightspeed Genomics, Inc. | Apparatus for selective excitation of microparticles |
WO2009052214A2 (en) | 2007-10-15 | 2009-04-23 | Complete Genomics, Inc. | Sequence analysis using decorated nucleic acids |
US7811810B2 (en) | 2007-10-25 | 2010-10-12 | Industrial Technology Research Institute | Bioassay system including optical detection apparatuses, and method for detecting biomolecules |
US7767441B2 (en) * | 2007-10-25 | 2010-08-03 | Industrial Technology Research Institute | Bioassay system including optical detection apparatuses, and method for detecting biomolecules |
US8415099B2 (en) | 2007-11-05 | 2013-04-09 | Complete Genomics, Inc. | Efficient base determination in sequencing reactions |
WO2009073629A2 (en) | 2007-11-29 | 2009-06-11 | Complete Genomics, Inc. | Efficient shotgun sequencing methods |
US8592150B2 (en) | 2007-12-05 | 2013-11-26 | Complete Genomics, Inc. | Methods and compositions for long fragment read sequencing |
US20090156412A1 (en) * | 2007-12-17 | 2009-06-18 | Helicos Biosciences Corporation | Surface-capture of target nucleic acids |
US20090171640A1 (en) * | 2007-12-28 | 2009-07-02 | Microsoft Corporation | Population sequencing using short read technologies |
US20090181390A1 (en) * | 2008-01-11 | 2009-07-16 | Signosis, Inc. A California Corporation | High throughput detection of micrornas and use for disease diagnosis |
WO2009097368A2 (en) | 2008-01-28 | 2009-08-06 | Complete Genomics, Inc. | Methods and compositions for efficient base calling in sequencing reactions |
US7767400B2 (en) * | 2008-02-03 | 2010-08-03 | Helicos Biosciences Corporation | Paired-end reads in sequencing by synthesis |
US8252911B2 (en) * | 2008-02-12 | 2012-08-28 | Pacific Biosciences Of California, Inc. | Compositions and methods for use in analytical reactions |
US20090226906A1 (en) * | 2008-03-05 | 2009-09-10 | Helicos Biosciences Corporation | Methods and compositions for reducing nucleotide impurities |
WO2009120372A2 (en) | 2008-03-28 | 2009-10-01 | Pacific Biosciences Of California, Inc. | Compositions and methods for nucleic acid sequencing |
AU2009246180B2 (en) | 2008-05-14 | 2015-11-05 | Dermtech International | Diagnosis of melanoma and solar lentigo by nucleic acid analysis |
US8470164B2 (en) | 2008-06-25 | 2013-06-25 | Life Technologies Corporation | Methods and apparatus for measuring analytes using large scale FET arrays |
EP4047367A1 (en) | 2008-07-18 | 2022-08-24 | Bio-Rad Laboratories, Inc. | Method for detecting target analytes with droplet libraries |
US8808986B2 (en) | 2008-08-27 | 2014-08-19 | Gen9, Inc. | Methods and devices for high fidelity polynucleotide synthesis |
US20100301398A1 (en) | 2009-05-29 | 2010-12-02 | Ion Torrent Systems Incorporated | Methods and apparatus for measuring analytes |
US20100137143A1 (en) | 2008-10-22 | 2010-06-03 | Ion Torrent Systems Incorporated | Methods and apparatus for measuring analytes |
KR20110138340A (en) * | 2009-01-20 | 2011-12-27 | 더 보드 어브 트러스티스 어브 더 리랜드 스탠포드 주니어 유니버시티 | Single cell gene expression for diagnosis, prognosis and identification of drug targets |
GB0904957D0 (en) | 2009-03-23 | 2009-05-06 | Univ Erasmus Medical Ct | Tumour gene profile |
EP3415235A1 (en) | 2009-03-23 | 2018-12-19 | Raindance Technologies Inc. | Manipulation of microfluidic droplets |
WO2010126614A2 (en) | 2009-04-30 | 2010-11-04 | Good Start Genetics, Inc. | Methods and compositions for evaluating genetic markers |
US8776573B2 (en) | 2009-05-29 | 2014-07-15 | Life Technologies Corporation | Methods and apparatus for measuring analytes |
US8673627B2 (en) | 2009-05-29 | 2014-03-18 | Life Technologies Corporation | Apparatus and methods for performing electrochemical reactions |
WO2010141390A2 (en) | 2009-06-05 | 2010-12-09 | Life Technologies Corporation | Nucleotide transient binding for sequencing methods |
US9524369B2 (en) | 2009-06-15 | 2016-12-20 | Complete Genomics, Inc. | Processing and analysis of complex nucleic acid sequence data |
US9416409B2 (en) | 2009-07-31 | 2016-08-16 | Ibis Biosciences, Inc. | Capture primers and capture sequence linked solid supports for molecular diagnostic tests |
CN102625838B (en) * | 2009-08-14 | 2016-01-20 | 阿霹震中科技公司 | For generating sample that rRNA eliminates or for from the method for sample separation rRNA, composition and test kit |
CA3047466A1 (en) | 2009-09-08 | 2011-03-17 | Laboratory Corporation Of America Holdings | Compositions and methods for diagnosing autism spectrum disorders |
US10174368B2 (en) | 2009-09-10 | 2019-01-08 | Centrillion Technology Holdings Corporation | Methods and systems for sequencing long nucleic acids |
WO2011032040A1 (en) | 2009-09-10 | 2011-03-17 | Centrillion Technology Holding Corporation | Methods of targeted sequencing |
KR20120100938A (en) | 2009-09-23 | 2012-09-12 | 셀매틱스, 인크. | Methods and devices for assessing infertility and/or egg quality |
WO2011042564A1 (en) | 2009-10-09 | 2011-04-14 | Universite De Strasbourg | Labelled silica-based nanomaterial with enhanced properties and uses thereof |
US8236570B2 (en) | 2009-11-03 | 2012-08-07 | Infoscitex | Methods for identifying nucleic acid ligands |
US10207240B2 (en) | 2009-11-03 | 2019-02-19 | Gen9, Inc. | Methods and microfluidic devices for the manipulation of droplets in high fidelity polynucleotide assembly |
CA2779750C (en) | 2009-11-06 | 2019-03-19 | The Board Of Trustees Of The Leland Stanford Junior University | Non-invasive diagnosis of graft rejection in organ transplant patients |
WO2011066185A1 (en) | 2009-11-25 | 2011-06-03 | Gen9, Inc. | Microfluidic devices and methods for gene synthesis |
EP2517025B1 (en) | 2009-12-23 | 2019-11-27 | Bio-Rad Laboratories, Inc. | Methods for reducing the exchange of molecules between droplets |
US9217144B2 (en) | 2010-01-07 | 2015-12-22 | Gen9, Inc. | Assembly of high fidelity polynucleotides |
US9399797B2 (en) | 2010-02-12 | 2016-07-26 | Raindance Technologies, Inc. | Digital analyte analysis |
US9366632B2 (en) | 2010-02-12 | 2016-06-14 | Raindance Technologies, Inc. | Digital analyte analysis |
EP2534267B1 (en) | 2010-02-12 | 2018-04-11 | Raindance Technologies, Inc. | Digital analyte analysis |
US10351905B2 (en) | 2010-02-12 | 2019-07-16 | Bio-Rad Laboratories, Inc. | Digital analyte analysis |
CA2790434A1 (en) | 2010-02-18 | 2011-08-25 | Anthony P. Shuber | Compositions and methods for treating cancer |
US9465228B2 (en) | 2010-03-19 | 2016-10-11 | Optical Biosystems, Inc. | Illumination apparatus optimized for synthetic aperture optics imaging using minimum selective excitation patterns |
US8502867B2 (en) | 2010-03-19 | 2013-08-06 | Lightspeed Genomics, Inc. | Synthetic aperture optics imaging method using minimum selective excitation patterns |
US9476812B2 (en) | 2010-04-21 | 2016-10-25 | Dna Electronics, Inc. | Methods for isolating a target analyte from a heterogeneous sample |
US20110262989A1 (en) | 2010-04-21 | 2011-10-27 | Nanomr, Inc. | Isolating a target analyte from a body fluid |
US8841104B2 (en) | 2010-04-21 | 2014-09-23 | Nanomr, Inc. | Methods for isolating a target analyte from a heterogeneous sample |
AU2011249913B2 (en) | 2010-05-06 | 2014-09-11 | Ibis Biosciences, Inc. | Integrated sample preparation systems and stabilized enzyme mixtures |
US9234240B2 (en) | 2010-05-07 | 2016-01-12 | The Board Of Trustees Of The Leland Stanford Junior University | Measurement and comparison of immune diversity by high-throughput sequencing |
US9670243B2 (en) * | 2010-06-02 | 2017-06-06 | Industrial Technology Research Institute | Compositions and methods for sequencing nucleic acids |
AU2011226767B1 (en) | 2010-06-30 | 2011-11-10 | Life Technologies Corporation | Ion-sensing charge-accumulation circuits and methods |
US8731847B2 (en) | 2010-06-30 | 2014-05-20 | Life Technologies Corporation | Array configuration and readout scheme |
JP5952813B2 (en) | 2010-06-30 | 2016-07-13 | ライフ テクノロジーズ コーポレーション | Method and apparatus for testing ISFET arrays |
EP2589065B1 (en) | 2010-07-03 | 2015-08-19 | Life Technologies Corporation | Chemically sensitive sensor with lightly doped drains |
CN103069006A (en) | 2010-07-23 | 2013-04-24 | 艾索特里克斯遗传实验室有限责任公司 | Identification of differentially represented fetal or maternal genomic regions and uses thereof |
WO2012018387A2 (en) | 2010-08-02 | 2012-02-09 | Population Diagnotics, Inc. | Compositions and methods for discovery of causative mutations in genetic disorders |
WO2012036679A1 (en) | 2010-09-15 | 2012-03-22 | Life Technologies Corporation | Methods and apparatus for measuring analytes |
AU2011301935B2 (en) | 2010-09-16 | 2015-06-11 | Ibis Biosciences, Inc. | Stabilization of ozone-labile fluorescent dyes by thiourea |
US9938590B2 (en) | 2010-09-16 | 2018-04-10 | Gen-Probe Incorporated | Capture probes immobilizable via L-nucleotide tail |
EP2619564B1 (en) | 2010-09-24 | 2016-03-16 | Life Technologies Corporation | Matched pair transistor circuits |
US9562897B2 (en) | 2010-09-30 | 2017-02-07 | Raindance Technologies, Inc. | Sandwich assays in droplets |
US9255293B2 (en) | 2010-11-01 | 2016-02-09 | Gen-Probe Incorporated | Integrated capture and amplification of target nucleic acid for sequencing |
US9074251B2 (en) * | 2011-02-10 | 2015-07-07 | Illumina, Inc. | Linking sequence reads using paired code tags |
EP3000883B8 (en) | 2010-11-12 | 2018-02-28 | Gen9, Inc. | Methods and devices for nucleic acids synthesis |
WO2012064975A1 (en) | 2010-11-12 | 2012-05-18 | Gen9, Inc. | Protein arrays and methods of using and making the same |
CN108744262A (en) | 2010-11-23 | 2018-11-06 | 普莱萨格生命科学公司 | Treatment and composition for for physical delivery |
US9163281B2 (en) | 2010-12-23 | 2015-10-20 | Good Start Genetics, Inc. | Methods for maintaining the integrity and identification of a nucleic acid template in a multiplex sequencing reaction |
CN105755545B (en) | 2010-12-27 | 2019-05-03 | 艾比斯生物科学公司 | The preparation method and composition of nucleic acid samples |
WO2012100194A1 (en) | 2011-01-20 | 2012-07-26 | Ibis Biosciences, Inc. | Microfluidic transducer |
CN103703143B (en) | 2011-01-31 | 2016-12-14 | 爱普瑞斯生物公司 | The method of the multiple epi-positions in identification of cell |
US9364803B2 (en) | 2011-02-11 | 2016-06-14 | Raindance Technologies, Inc. | Methods for forming mixed droplets |
US9150852B2 (en) | 2011-02-18 | 2015-10-06 | Raindance Technologies, Inc. | Compositions and methods for molecular labeling |
KR20190006083A (en) | 2011-03-09 | 2019-01-16 | 셀 시그널링 테크놀러지, 인크. | Methods and reagents for creating monoclonal antibodies |
US20120252682A1 (en) | 2011-04-01 | 2012-10-04 | Maples Corporate Services Limited | Methods and systems for sequencing nucleic acids |
EP3709018A1 (en) | 2011-06-02 | 2020-09-16 | Bio-Rad Laboratories, Inc. | Microfluidic apparatus for identifying components of a chemical reaction |
US8841071B2 (en) | 2011-06-02 | 2014-09-23 | Raindance Technologies, Inc. | Sample multiplexing |
US9752176B2 (en) | 2011-06-15 | 2017-09-05 | Ginkgo Bioworks, Inc. | Methods for preparative in vitro cloning |
US20140235474A1 (en) | 2011-06-24 | 2014-08-21 | Sequenom, Inc. | Methods and processes for non invasive assessment of a genetic variation |
US8658430B2 (en) | 2011-07-20 | 2014-02-25 | Raindance Technologies, Inc. | Manipulating droplet size |
WO2013022778A1 (en) | 2011-08-05 | 2013-02-14 | Ibis Biosciences, Inc. | Nucleic acid sequencing by electrochemical detection |
DK3594340T3 (en) | 2011-08-26 | 2021-09-20 | Gen9 Inc | COMPOSITIONS AND METHODS FOR COLLECTING WITH HIGH ACCURACY OF NUCLEIC ACIDS |
SG11201401222WA (en) | 2011-10-03 | 2014-09-26 | Celmatix Inc | Methods and devices for assessing risk to a putative offspring of developing a condition |
US9984198B2 (en) | 2011-10-06 | 2018-05-29 | Sequenom, Inc. | Reducing sequence read count error in assessment of complex genetic variations |
US10424394B2 (en) | 2011-10-06 | 2019-09-24 | Sequenom, Inc. | Methods and processes for non-invasive assessment of genetic variations |
US10196681B2 (en) | 2011-10-06 | 2019-02-05 | Sequenom, Inc. | Methods and processes for non-invasive assessment of genetic variations |
CA2850785C (en) | 2011-10-06 | 2022-12-13 | Sequenom, Inc. | Methods and processes for non-invasive assessment of genetic variations |
US9367663B2 (en) | 2011-10-06 | 2016-06-14 | Sequenom, Inc. | Methods and processes for non-invasive assessment of genetic variations |
CA2851388C (en) | 2011-10-10 | 2023-11-21 | The Hospital For Sick Children | Methods and compositions for screening and treating developmental disorders |
US9228233B2 (en) | 2011-10-17 | 2016-01-05 | Good Start Genetics, Inc. | Analysis methods |
EP2769007B1 (en) | 2011-10-19 | 2016-12-07 | Nugen Technologies, Inc. | Compositions and methods for directional nucleic acid amplification and sequencing |
WO2013063308A1 (en) | 2011-10-25 | 2013-05-02 | University Of Massachusetts | An enzymatic method to enrich for capped rna, kits for performing same, and compositions derived therefrom |
US11180807B2 (en) | 2011-11-04 | 2021-11-23 | Population Bio, Inc. | Methods for detecting a genetic variation in attractin-like 1 (ATRNL1) gene in subject with Parkinson's disease |
CA2854832C (en) | 2011-11-07 | 2023-05-23 | Ingenuity Systems, Inc. | Methods and systems for identification of causal genomic variants |
GB201120711D0 (en) | 2011-12-01 | 2012-01-11 | Univ Erasmus Medical Ct | Method for classifying tumour cells |
US9970984B2 (en) | 2011-12-01 | 2018-05-15 | Life Technologies Corporation | Method and apparatus for identifying defects in a chemical sensor array |
AU2012352965B2 (en) * | 2011-12-16 | 2014-08-21 | Li-Cor, Inc. | Luminescence imaging scanner |
US9803188B2 (en) | 2011-12-22 | 2017-10-31 | Ibis Biosciences, Inc. | Systems and methods for isolating nucleic acids |
EP2794927B1 (en) | 2011-12-22 | 2017-04-12 | Ibis Biosciences, Inc. | Amplification primers and methods |
US10150993B2 (en) | 2011-12-22 | 2018-12-11 | Ibis Biosciences, Inc. | Macromolecule positioning by electrical potential |
EP3269820A1 (en) | 2011-12-22 | 2018-01-17 | Ibis Biosciences, Inc. | Kit for the amplification of a sequence from a ribonucleic acid |
US9334491B2 (en) | 2011-12-22 | 2016-05-10 | Ibis Biosciences, Inc. | Systems and methods for isolating nucleic acids from cellular samples |
WO2013102091A1 (en) | 2011-12-28 | 2013-07-04 | Ibis Biosciences, Inc. | Nucleic acid ligation systems and methods |
WO2013102081A2 (en) | 2011-12-29 | 2013-07-04 | Ibis Biosciences, Inc. | Macromolecule delivery to nanowells |
JP2015503923A (en) | 2012-01-09 | 2015-02-05 | オスロ ウニヴェルスィテーツスィーケフース ハーエフOslo Universitetssykehus Hf | Methods and biomarkers for the analysis of colorectal cancer |
WO2013106737A1 (en) | 2012-01-13 | 2013-07-18 | Data2Bio | Genotyping by next-generation sequencing |
US8747748B2 (en) | 2012-01-19 | 2014-06-10 | Life Technologies Corporation | Chemical sensor with conductive cup-shaped sensor surface |
US8821798B2 (en) | 2012-01-19 | 2014-09-02 | Life Technologies Corporation | Titanium nitride as sensing layer for microwell structure |
WO2013109981A1 (en) | 2012-01-20 | 2013-07-25 | Sequenom, Inc. | Diagnostic processes that factor experimental conditions |
WO2013112923A1 (en) | 2012-01-26 | 2013-08-01 | Nugen Technologies, Inc. | Compositions and methods for targeted nucleic acid sequence enrichment and high efficiency library generation |
WO2013116774A1 (en) | 2012-02-01 | 2013-08-08 | Gen-Probe Incorporated | Asymmetric hairpin target capture oligomers |
WO2013120018A1 (en) | 2012-02-09 | 2013-08-15 | Population Diagnostics, Inc. | Methods and compositions for screening and treating developmental disorders |
WO2013120089A1 (en) | 2012-02-10 | 2013-08-15 | Raindance Technologies, Inc. | Molecular diagnostic screening assay |
EP3363901B1 (en) | 2012-02-17 | 2020-12-30 | Fred Hutchinson Cancer Research Center | Compositions and methods for accurately identifying mutations |
US9176031B2 (en) | 2012-02-24 | 2015-11-03 | Raindance Technologies, Inc. | Labeling and sample preparation for sequencing |
EP2820174B1 (en) | 2012-02-27 | 2019-12-25 | The University of North Carolina at Chapel Hill | Methods and uses for molecular tags |
US9150853B2 (en) | 2012-03-21 | 2015-10-06 | Gen9, Inc. | Methods for screening proteins using DNA encoded chemical libraries as templates for enzyme catalysis |
EP2834370B1 (en) | 2012-04-03 | 2019-01-02 | The Regents Of The University Of Michigan | Biomarker associated with irritable bowel syndrome and crohn's disease |
US8209130B1 (en) | 2012-04-04 | 2012-06-26 | Good Start Genetics, Inc. | Sequence assembly |
US8812422B2 (en) | 2012-04-09 | 2014-08-19 | Good Start Genetics, Inc. | Variant database |
US10227635B2 (en) | 2012-04-16 | 2019-03-12 | Molecular Loop Biosolutions, Llc | Capture reactions |
EP3543350B1 (en) | 2012-04-24 | 2021-11-10 | Gen9, Inc. | Methods for sorting nucleic acids and multiplexed preparative in vitro cloning |
WO2013165748A1 (en) | 2012-04-30 | 2013-11-07 | Raindance Technologies, Inc | Digital analyte analysis |
US20150133310A1 (en) | 2012-05-02 | 2015-05-14 | Ibis Biosciences, Inc. | Nucleic acid sequencing systems and methods |
ES2683707T3 (en) | 2012-05-02 | 2018-09-27 | Ibis Biosciences, Inc. | DNA sequencing |
US9512477B2 (en) | 2012-05-04 | 2016-12-06 | Boreal Genomics Inc. | Biomarker anaylsis using scodaphoresis |
JP6445426B2 (en) | 2012-05-10 | 2018-12-26 | ザ ジェネラル ホスピタル コーポレイション | Method for determining nucleotide sequence |
US9920361B2 (en) | 2012-05-21 | 2018-03-20 | Sequenom, Inc. | Methods and compositions for analyzing nucleic acid |
US10504613B2 (en) | 2012-12-20 | 2019-12-10 | Sequenom, Inc. | Methods and processes for non-invasive assessment of genetic variations |
AU2012380717B2 (en) | 2012-05-24 | 2018-08-16 | Fundacio Institucio Catalana De Recerca I Estudis Avancats | Method for the identification of the origin of a cancer of unknown primary origin by methylation analysis |
US8786331B2 (en) | 2012-05-29 | 2014-07-22 | Life Technologies Corporation | System for reducing noise in a chemical sensor array |
WO2013186306A1 (en) | 2012-06-15 | 2013-12-19 | Boehringer Ingelheim International Gmbh | Method for identifying transcriptional regulatory elements |
CA2877094A1 (en) | 2012-06-18 | 2013-12-27 | Nugen Technologies, Inc. | Compositions and methods for negative selection of non-desired nucleic acid sequences |
US10497461B2 (en) | 2012-06-22 | 2019-12-03 | Sequenom, Inc. | Methods and processes for non-invasive assessment of genetic variations |
IL236303B (en) | 2012-06-25 | 2022-07-01 | Gen9 Inc | Methods for nucleic acid assembly and high throughput sequencing |
WO2014005076A2 (en) | 2012-06-29 | 2014-01-03 | The Regents Of The University Of Michigan | Methods and biomarkers for detection of kidney disorders |
US20150011396A1 (en) | 2012-07-09 | 2015-01-08 | Benjamin G. Schroeder | Methods for creating directional bisulfite-converted nucleic acid libraries for next generation sequencing |
EP2882868B1 (en) | 2012-08-08 | 2019-07-31 | H. Hoffnabb-La Roche Ag | Increasing dynamic range for identifying multiple epitopes in cells |
EP2895622A4 (en) | 2012-09-11 | 2016-05-18 | Theranos Inc | Information management systems and methods using a biological signature |
DK2895621T3 (en) | 2012-09-14 | 2020-11-30 | Population Bio Inc | METHODS AND COMPOSITION FOR DIAGNOSIS, FORECAST AND TREATMENT OF NEUROLOGICAL CONDITIONS |
CA2922005A1 (en) | 2012-09-27 | 2014-04-03 | Population Diagnostics, Inc. | Methods and compositions for screening and treating developmental disorders |
US10482994B2 (en) | 2012-10-04 | 2019-11-19 | Sequenom, Inc. | Methods and processes for non-invasive assessment of genetic variations |
ES2701750T3 (en) | 2012-10-16 | 2019-02-25 | Abbott Molecular Inc | Procedures for sequencing a nucleic acid |
US10162800B2 (en) | 2012-10-17 | 2018-12-25 | Celmatix Inc. | Systems and methods for determining the probability of a pregnancy at a selected point in time |
US9177098B2 (en) | 2012-10-17 | 2015-11-03 | Celmatix Inc. | Systems and methods for determining the probability of a pregnancy at a selected point in time |
US9836577B2 (en) | 2012-12-14 | 2017-12-05 | Celmatix, Inc. | Methods and devices for assessing risk of female infertility |
US9804069B2 (en) | 2012-12-19 | 2017-10-31 | Dnae Group Holdings Limited | Methods for degrading nucleic acid |
US9995742B2 (en) | 2012-12-19 | 2018-06-12 | Dnae Group Holdings Limited | Sample entry |
US9434940B2 (en) | 2012-12-19 | 2016-09-06 | Dna Electronics, Inc. | Methods for universal target capture |
US9599610B2 (en) | 2012-12-19 | 2017-03-21 | Dnae Group Holdings Limited | Target capture system |
US9551704B2 (en) | 2012-12-19 | 2017-01-24 | Dna Electronics, Inc. | Target detection |
US10000557B2 (en) | 2012-12-19 | 2018-06-19 | Dnae Group Holdings Limited | Methods for raising antibodies |
US9080968B2 (en) | 2013-01-04 | 2015-07-14 | Life Technologies Corporation | Methods and systems for point of use removal of sacrificial material |
US9841398B2 (en) | 2013-01-08 | 2017-12-12 | Life Technologies Corporation | Methods for manufacturing well structures for low-noise chemical sensors |
CN105190656B (en) | 2013-01-17 | 2018-01-16 | 佩索纳里斯公司 | Method and system for genetic analysis |
WO2014116729A2 (en) | 2013-01-22 | 2014-07-31 | The Board Of Trustees Of The Leland Stanford Junior University | Haplotying of hla loci with ultra-deep shotgun sequencing |
US20130309666A1 (en) | 2013-01-25 | 2013-11-21 | Sequenom, Inc. | Methods and processes for non-invasive assessment of genetic variations |
US8962366B2 (en) | 2013-01-28 | 2015-02-24 | Life Technologies Corporation | Self-aligned well structures for low-noise chemical sensors |
US9411930B2 (en) | 2013-02-01 | 2016-08-09 | The Regents Of The University Of California | Methods for genome assembly and haplotype phasing |
GB2547875B (en) | 2013-02-01 | 2017-12-13 | Univ California | Methods for meta-genomics analysis of microbes |
US9303263B2 (en) | 2013-03-01 | 2016-04-05 | Vivonics, Inc. | Aptamers that bind CD271 |
US8963216B2 (en) | 2013-03-13 | 2015-02-24 | Life Technologies Corporation | Chemical sensor with sidewall spacer sensor surface |
US8841217B1 (en) | 2013-03-13 | 2014-09-23 | Life Technologies Corporation | Chemical sensor with protruded sensor surface |
US9701999B2 (en) | 2013-03-14 | 2017-07-11 | Abbott Molecular, Inc. | Multiplex methylation-specific amplification systems and methods |
EP2971154A4 (en) | 2013-03-14 | 2017-03-01 | Ibis Biosciences, Inc. | Nucleic acid control panels |
US9146248B2 (en) | 2013-03-14 | 2015-09-29 | Intelligent Bio-Systems, Inc. | Apparatus and methods for purging flow cells in nucleic acid sequencing instruments |
EP2971159B1 (en) | 2013-03-14 | 2019-05-08 | Molecular Loop Biosolutions, LLC | Methods for analyzing nucleic acids |
US9591268B2 (en) | 2013-03-15 | 2017-03-07 | Qiagen Waltham, Inc. | Flow cell alignment methods and systems |
CN105283758B (en) | 2013-03-15 | 2018-06-05 | 生命科技公司 | Chemical sensor with consistent sensor surface area |
WO2014144092A1 (en) | 2013-03-15 | 2014-09-18 | Nugen Technologies, Inc. | Sequential sequencing |
CN105264366B (en) | 2013-03-15 | 2019-04-16 | 生命科技公司 | Chemical sensor with consistent sensor surface area |
CN105358709B (en) | 2013-03-15 | 2018-12-07 | 雅培分子公司 | System and method for detecting genome copy numbers variation |
DK3327123T3 (en) | 2013-03-15 | 2019-11-25 | Lineage Biosciences Inc | METHODS FOR SEQUENCING THE IMMUN REPERTOIR |
EP3533884A1 (en) | 2013-03-15 | 2019-09-04 | Ibis Biosciences, Inc. | Dna sequences to assess contamination in dna sequencing |
CA2906076A1 (en) | 2013-03-15 | 2014-09-18 | Abvitro, Inc. | Single cell bar-coding for antibody discovery |
US9116117B2 (en) | 2013-03-15 | 2015-08-25 | Life Technologies Corporation | Chemical sensor with sidewall sensor surface |
EP2981921B1 (en) | 2013-04-03 | 2023-01-18 | Sequenom, Inc. | Methods and processes for non-invasive assessment of genetic variations |
EP2986762B1 (en) | 2013-04-19 | 2019-11-06 | Bio-Rad Laboratories, Inc. | Digital analyte analysis |
US9725724B2 (en) | 2013-05-16 | 2017-08-08 | Vivonics, Inc. | Neutral nucleic acid ligands |
IL309903A (en) | 2013-05-24 | 2024-03-01 | Sequenom Inc | Methods and processes for non-invasive assessment of genetic variations |
US8847799B1 (en) | 2013-06-03 | 2014-09-30 | Good Start Genetics, Inc. | Methods and systems for storing sequence read data |
US10458942B2 (en) | 2013-06-10 | 2019-10-29 | Life Technologies Corporation | Chemical sensor array having multiple sensors per well |
EP3540076A1 (en) | 2013-06-21 | 2019-09-18 | Sequenom, Inc. | Methods and processes for non-invasive assessment of genetic variations |
DK3030682T3 (en) | 2013-08-05 | 2020-09-14 | Twist Bioscience Corp | DE NOVO SYNTHESIZED GENE LIBRARIES |
EP3036359B1 (en) | 2013-08-19 | 2019-10-23 | Abbott Molecular Inc. | Next-generation sequencing libraries |
US9898575B2 (en) | 2013-08-21 | 2018-02-20 | Seven Bridges Genomics Inc. | Methods and systems for aligning sequences |
US9116866B2 (en) | 2013-08-21 | 2015-08-25 | Seven Bridges Genomics Inc. | Methods and systems for detecting sequence variants |
EP3039161B1 (en) | 2013-08-30 | 2021-10-06 | Personalis, Inc. | Methods and systems for genomic analysis |
EP3053073B1 (en) | 2013-09-30 | 2019-07-03 | Seven Bridges Genomics Inc. | Methods and system for detecting sequence variants |
WO2015051275A1 (en) | 2013-10-03 | 2015-04-09 | Personalis, Inc. | Methods for analyzing genotypes |
US11901041B2 (en) | 2013-10-04 | 2024-02-13 | Bio-Rad Laboratories, Inc. | Digital analysis of nucleic acid modification |
IL295860B2 (en) | 2013-10-04 | 2024-01-01 | Sequenom Inc | Methods and processes for non-invasive assessment of genetic variations |
JP6680680B2 (en) | 2013-10-07 | 2020-04-15 | セクエノム, インコーポレイテッド | Methods and processes for non-invasive assessment of chromosomal alterations |
CN105849279B (en) | 2013-10-18 | 2020-02-18 | 七桥基因公司 | Methods and systems for identifying disease-induced mutations |
US10851414B2 (en) | 2013-10-18 | 2020-12-01 | Good Start Genetics, Inc. | Methods for determining carrier status |
WO2015058120A1 (en) | 2013-10-18 | 2015-04-23 | Seven Bridges Genomics Inc. | Methods and systems for aligning sequences in the presence of repeating elements |
US10832797B2 (en) | 2013-10-18 | 2020-11-10 | Seven Bridges Genomics Inc. | Method and system for quantifying sequence alignment |
WO2015057565A1 (en) | 2013-10-18 | 2015-04-23 | Good Start Genetics, Inc. | Methods for assessing a genomic region of a subject |
EP3058332B1 (en) | 2013-10-18 | 2019-08-28 | Seven Bridges Genomics Inc. | Methods and systems for genotyping genetic samples |
US9092402B2 (en) | 2013-10-21 | 2015-07-28 | Seven Bridges Genomics Inc. | Systems and methods for using paired-end data in directed acyclic structure |
BR112016010095A2 (en) | 2013-11-07 | 2017-09-12 | Univ Leland Stanford Junior | free cell nucleic acids for human microbiome analysis and components thereof. |
EP2891722B1 (en) | 2013-11-12 | 2018-10-10 | Population Bio, Inc. | Methods and compositions for diagnosing, prognosing, and treating endometriosis |
EP3068883B1 (en) | 2013-11-13 | 2020-04-29 | Nugen Technologies, Inc. | Compositions and methods for identification of a duplicate sequencing read |
US9546398B2 (en) * | 2013-11-14 | 2017-01-17 | Agilent Technologies, Inc. | Polymerase idling method for single molecule DNA sequencing |
AU2014348306B2 (en) | 2013-11-17 | 2020-11-12 | Quantum-Si Incorporated | Active-source-pixel, integrated device for rapid analysis of biological and chemical specimens |
WO2015089243A1 (en) | 2013-12-11 | 2015-06-18 | The Regents For Of The University Of California | Methods for labeling dna fragments to recontruct physical linkage and phase |
US9944977B2 (en) | 2013-12-12 | 2018-04-17 | Raindance Technologies, Inc. | Distinguishing rare variations in a nucleic acid sequence from a sample |
WO2015095355A2 (en) | 2013-12-17 | 2015-06-25 | The Brigham And Women's Hospital, Inc. | Detection of an antibody against a pathogen |
DE202014010499U1 (en) | 2013-12-17 | 2015-10-20 | Kymab Limited | Targeting of human PCSK9 for cholesterol treatment |
US11193176B2 (en) | 2013-12-31 | 2021-12-07 | Bio-Rad Laboratories, Inc. | Method for detecting and quantifying latent retroviral RNA species |
WO2015105963A1 (en) | 2014-01-10 | 2015-07-16 | Seven Bridges Genomics Inc. | Systems and methods for use of known alleles in read mapping |
WO2015107430A2 (en) | 2014-01-16 | 2015-07-23 | Oslo Universitetssykehus Hf | Methods and biomarkers for detection and prognosis of cervical cancer |
WO2015112974A1 (en) | 2014-01-27 | 2015-07-30 | The General Hospital Corporation | Methods of preparing nucleic acids for sequencing |
US9817944B2 (en) | 2014-02-11 | 2017-11-14 | Seven Bridges Genomics Inc. | Systems and methods for analyzing sequence data |
WO2015131107A1 (en) | 2014-02-28 | 2015-09-03 | Nugen Technologies, Inc. | Reduced representation bisulfite sequencing with diversity adaptors |
WO2015175530A1 (en) | 2014-05-12 | 2015-11-19 | Gore Athurva | Methods for detecting aneuploidy |
US10760109B2 (en) | 2014-06-06 | 2020-09-01 | The Regents Of The University Of Michigan | Compositions and methods for characterizing and diagnosing periodontal disease |
WO2015200378A1 (en) | 2014-06-23 | 2015-12-30 | The General Hospital Corporation | Genomewide unbiased identification of dsbs evaluated by sequencing (guide-seq) |
CN114214314A (en) | 2014-06-24 | 2022-03-22 | 生物辐射实验室股份有限公司 | Digital PCR barcoding |
DE202015009002U1 (en) | 2014-07-15 | 2016-08-18 | Kymab Limited | Targeting of human PCSK9 for cholesterol treatment |
EP3332790A1 (en) | 2014-07-15 | 2018-06-13 | Kymab Limited | Antibodies for use in treating conditions related to specific pcsk9 variants in specific patients populations |
WO2016008899A1 (en) | 2014-07-15 | 2016-01-21 | Kymab Limited | Targeting human pcsk9 for cholesterol treatment |
US10208350B2 (en) | 2014-07-17 | 2019-02-19 | Celmatix Inc. | Methods and systems for assessing infertility and related pathologies |
MX2017001134A (en) | 2014-07-24 | 2017-10-11 | Abbott Molecular Inc | Compositions and methods for the detection and analysis of mycobacterium tuberculosis. |
US11783911B2 (en) | 2014-07-30 | 2023-10-10 | Sequenom, Inc | Methods and processes for non-invasive assessment of genetic variations |
EP4219710A3 (en) | 2014-08-01 | 2023-08-16 | Dovetail Genomics, LLC | Tagging nucleic acids for sequence assembly |
SG11201700891SA (en) | 2014-08-06 | 2017-03-30 | Nugen Technologies Inc | Digital measurements from targeted sequencing |
CN112903638A (en) | 2014-08-08 | 2021-06-04 | 宽腾矽公司 | Integrated device with external light source for detection, detection and analysis of molecules |
EP3194933A1 (en) | 2014-08-08 | 2017-07-26 | Quantum-si Incorporated | Optical system and assay chip for probing, detecting, and analyzing molecules |
EP3194935B1 (en) | 2014-08-08 | 2018-10-31 | Quantum-si Incorporated | Integrated device for temporal binning of received photons |
WO2016023916A1 (en) | 2014-08-12 | 2016-02-18 | Kymab Limited | Treatment of disease using ligand binding to targets of interest |
GB2558326B (en) | 2014-09-05 | 2021-01-20 | Population Bio Inc | Methods and compositions for inhibiting and treating neurological conditions |
US9323569B2 (en) * | 2014-09-10 | 2016-04-26 | Amazon Technologies, Inc. | Scalable log-based transaction management |
US9519674B2 (en) * | 2014-09-10 | 2016-12-13 | Amazon Technologies, Inc. | Stateless datastore-independent transactions |
WO2016040446A1 (en) | 2014-09-10 | 2016-03-17 | Good Start Genetics, Inc. | Methods for selectively suppressing non-target sequences |
EP3950944A1 (en) | 2014-09-15 | 2022-02-09 | AbVitro LLC | High-throughput nucleotide library sequencing |
US10429399B2 (en) | 2014-09-24 | 2019-10-01 | Good Start Genetics, Inc. | Process control for increased robustness of genetic assays |
CA2964349C (en) | 2014-10-14 | 2023-03-21 | Seven Bridges Genomics Inc. | Systems and methods for smart tools in sequence pipelines |
EP4026913A1 (en) | 2014-10-30 | 2022-07-13 | Personalis, Inc. | Methods for using mosaicism in nucleic acids sampled distal to their origin |
US10000799B2 (en) | 2014-11-04 | 2018-06-19 | Boreal Genomics, Inc. | Methods of sequencing with linked fragments |
GB201419731D0 (en) * | 2014-11-05 | 2014-12-17 | Illumina Cambridge Ltd | Sequencing from multiple primers to increase data rate and density |
WO2016071701A1 (en) | 2014-11-07 | 2016-05-12 | Kymab Limited | Treatment of disease using ligand binding to targets of interest |
JP6767978B2 (en) | 2014-12-03 | 2020-10-14 | アイソプレキシス コーポレイション | Analysis and screening of cell secretion profiles |
US10077472B2 (en) | 2014-12-18 | 2018-09-18 | Life Technologies Corporation | High data rate integrated circuit with power management |
EP4095261A1 (en) | 2015-01-06 | 2022-11-30 | Molecular Loop Biosciences, Inc. | Screening for structural variants |
US9984201B2 (en) | 2015-01-18 | 2018-05-29 | Youhealth Biotech, Limited | Method and system for determining cancer status |
WO2016126987A1 (en) | 2015-02-04 | 2016-08-11 | Twist Bioscience Corporation | Compositions and methods for synthetic gene assembly |
US10669304B2 (en) | 2015-02-04 | 2020-06-02 | Twist Bioscience Corporation | Methods and devices for de novo oligonucleic acid assembly |
WO2016134034A1 (en) | 2015-02-17 | 2016-08-25 | Dovetail Genomics Llc | Nucleic acid sequence assembly |
US10208339B2 (en) | 2015-02-19 | 2019-02-19 | Takara Bio Usa, Inc. | Systems and methods for whole genome amplification |
EP3822361A1 (en) | 2015-02-20 | 2021-05-19 | Takara Bio USA, Inc. | Method for rapid accurate dispensing, visualization and analysis of single cells |
US10192026B2 (en) | 2015-03-05 | 2019-01-29 | Seven Bridges Genomics Inc. | Systems and methods for genomic pattern analysis |
US11807896B2 (en) | 2015-03-26 | 2023-11-07 | Dovetail Genomics, Llc | Physical linkage preservation in DNA storage |
JP2018518155A (en) | 2015-04-15 | 2018-07-12 | ザ ジェネラル ホスピタル コーポレイション | LNA-based mutant enrichment next generation sequencing assay |
WO2016172377A1 (en) | 2015-04-21 | 2016-10-27 | Twist Bioscience Corporation | Devices and methods for oligonucleic acid library synthesis |
US10275567B2 (en) | 2015-05-22 | 2019-04-30 | Seven Bridges Genomics Inc. | Systems and methods for haplotyping |
US10392613B2 (en) | 2015-07-14 | 2019-08-27 | Abbott Molecular Inc. | Purification of nucleic acids using copper-titanium oxides |
US10526664B2 (en) | 2015-07-14 | 2020-01-07 | Abbott Molecular Inc. | Compositions and methods for identifying drug resistant tuberculosis |
US10793895B2 (en) | 2015-08-24 | 2020-10-06 | Seven Bridges Genomics Inc. | Systems and methods for epigenetic analysis |
US10724110B2 (en) | 2015-09-01 | 2020-07-28 | Seven Bridges Genomics Inc. | Systems and methods for analyzing viral nucleic acids |
US10584380B2 (en) | 2015-09-01 | 2020-03-10 | Seven Bridges Genomics Inc. | Systems and methods for mitochondrial analysis |
US10647981B1 (en) | 2015-09-08 | 2020-05-12 | Bio-Rad Laboratories, Inc. | Nucleic acid library generation methods and compositions |
AU2016319110B2 (en) | 2015-09-11 | 2022-01-27 | The General Hospital Corporation | Full interrogation of nuclease DSBs and sequencing (FIND-seq) |
WO2017049231A1 (en) | 2015-09-18 | 2017-03-23 | Twist Bioscience Corporation | Oligonucleic acid variant libraries and synthesis thereof |
US11512347B2 (en) | 2015-09-22 | 2022-11-29 | Twist Bioscience Corporation | Flexible substrates for nucleic acid synthesis |
EP3353325B1 (en) | 2015-09-24 | 2024-03-20 | AbVitro LLC | Single amplicon activated exclusion pcr |
CA2999888A1 (en) | 2015-09-24 | 2017-03-30 | Abvitro Llc | Affinity-oligonucleotide conjugates and uses thereof |
US10928392B2 (en) | 2015-09-25 | 2021-02-23 | Abvitro Llc | High throughput process for T cell receptor target identification of natively-paired T cell receptor sequences |
JP2018529353A (en) | 2015-09-30 | 2018-10-11 | ザ ジェネラル ホスピタル コーポレイション | Comprehensive in vitro reporting of cleavage events by sequencing (CIRCLE-seq) |
US11347704B2 (en) | 2015-10-16 | 2022-05-31 | Seven Bridges Genomics Inc. | Biological graph or sequence serialization |
CN108368542B (en) | 2015-10-19 | 2022-04-08 | 多弗泰尔基因组学有限责任公司 | Methods for genome assembly, haplotype phasing, and target-independent nucleic acid detection |
CA3006867A1 (en) | 2015-12-01 | 2017-06-08 | Twist Bioscience Corporation | Functionalized surfaces and preparation thereof |
US20170199960A1 (en) | 2016-01-07 | 2017-07-13 | Seven Bridges Genomics Inc. | Systems and methods for adaptive local alignment for graph genomes |
CN108431223A (en) | 2016-01-08 | 2018-08-21 | 生物辐射实验室股份有限公司 | Multiple pearls under per drop resolution |
US10364468B2 (en) | 2016-01-13 | 2019-07-30 | Seven Bridges Genomics Inc. | Systems and methods for analyzing circulating tumor DNA |
EP4012416A1 (en) | 2016-01-22 | 2022-06-15 | Purdue Research Foundation | Use of a charged mass labeling system for the detection of target analytes |
US10460829B2 (en) | 2016-01-26 | 2019-10-29 | Seven Bridges Genomics Inc. | Systems and methods for encoding genetic variation for a population |
WO2017139260A1 (en) | 2016-02-08 | 2017-08-17 | RGENE, Inc. | Multiple ligase compositions, systems, and methods |
US10519498B2 (en) | 2016-02-11 | 2019-12-31 | Qiagen Sciences, Llc | Additives to improve sequencing by synthesis performance |
EP3420108A4 (en) | 2016-02-23 | 2019-11-06 | Dovetail Genomics LLC | Generation of phased read-sets for genome assembly and haplotype phasing |
US10262102B2 (en) | 2016-02-24 | 2019-04-16 | Seven Bridges Genomics Inc. | Systems and methods for genotyping with graph reference |
EP3430154B1 (en) | 2016-03-14 | 2020-11-11 | Rgene, Inc. | Hyper-thermostable lysine-mutant ssdna/rna ligases |
KR102326769B1 (en) | 2016-03-25 | 2021-11-17 | 카리우스, 인코포레이티드 | Synthetic nucleic acid spike-ins |
EP3436607B1 (en) | 2016-03-28 | 2023-06-14 | Ncan Genomics, Inc. | Linked duplex target capture |
US10961573B2 (en) | 2016-03-28 | 2021-03-30 | Boreal Genomics, Inc. | Linked duplex target capture |
US11355328B2 (en) | 2016-04-13 | 2022-06-07 | Purdue Research Foundation | Systems and methods for isolating a target ion in an ion trap using a dual frequency waveform |
IL262946B2 (en) | 2016-05-13 | 2023-03-01 | Dovetail Genomics Llc | Recovering long-range linkage information from preserved samples |
ES2929367T3 (en) | 2016-05-18 | 2022-11-28 | Hoffmann La Roche | Quantitative ultrafast PCR amplification using an electrowet based device |
US11299783B2 (en) | 2016-05-27 | 2022-04-12 | Personalis, Inc. | Methods and systems for genetic analysis |
EP3464642A4 (en) | 2016-05-31 | 2020-02-19 | The Regents of the University of California | Methods for evaluating, monitoring, and modulating aging process |
US11624064B2 (en) | 2016-06-13 | 2023-04-11 | Grail, Llc | Enrichment of mutated cell free nucleic acids for cancer detection |
US11396678B2 (en) | 2016-07-06 | 2022-07-26 | The Regent Of The University Of California | Breast and ovarian cancer methylation markers and uses thereof |
US10093986B2 (en) | 2016-07-06 | 2018-10-09 | Youhealth Biotech, Limited | Leukemia methylation markers and uses thereof |
WO2018009707A1 (en) | 2016-07-06 | 2018-01-11 | Youhealth Biotech, Limited | Solid tumor methylation markers and uses thereof |
US11091795B2 (en) | 2016-07-11 | 2021-08-17 | Arizona Board Of Regents On Behalf Of The University Of Arizona | Compositions and methods for diagnosing and treating arrhythmias |
WO2018013558A1 (en) | 2016-07-12 | 2018-01-18 | Life Technologies Corporation | Compositions and methods for detecting nucleic acid regions |
CA3020629A1 (en) | 2016-07-21 | 2018-01-25 | Takara Bio Usa, Inc. | Multi-z imaging and dispensing with multi-well devices |
WO2018022890A1 (en) | 2016-07-27 | 2018-02-01 | Sequenom, Inc. | Genetic copy number alteration classifications |
EP3500672A4 (en) | 2016-08-22 | 2020-05-20 | Twist Bioscience Corporation | De novo synthesized nucleic acid libraries |
WO2018042251A1 (en) | 2016-08-29 | 2018-03-08 | Oslo Universitetssykehus Hf | Chip-seq assays |
US11250931B2 (en) | 2016-09-01 | 2022-02-15 | Seven Bridges Genomics Inc. | Systems and methods for detecting recombination |
CA3037190A1 (en) | 2016-09-15 | 2018-03-22 | ArcherDX, Inc. | Methods of nucleic acid sample preparation for analysis of cell-free dna |
AU2017328950B2 (en) | 2016-09-15 | 2023-09-14 | Archerdx, Llc | Methods of nucleic acid sample preparation |
US10417457B2 (en) | 2016-09-21 | 2019-09-17 | Twist Bioscience Corporation | Nucleic acid based data storage |
WO2018057928A1 (en) | 2016-09-23 | 2018-03-29 | Grail, Inc. | Methods of preparing and analyzing cell-free nucleic acid sequencing libraries |
JP6929354B2 (en) | 2016-09-24 | 2021-09-01 | アブビトロ, エルエルシー | Affinity-oligonucleotide conjugates and their use |
US10190155B2 (en) | 2016-10-14 | 2019-01-29 | Nugen Technologies, Inc. | Molecular tag attachment and transfer |
US11725232B2 (en) | 2016-10-31 | 2023-08-15 | The Hong Kong University Of Science And Technology | Compositions, methods and kits for detection of genetic variants for alzheimer's disease |
JP7161991B2 (en) | 2016-11-02 | 2022-10-27 | アーチャーディーエックス, エルエルシー | Methods of Nucleic Acid Sample Preparation for Immune Repertoire Sequencing |
CA3040930A1 (en) | 2016-11-07 | 2018-05-11 | Grail, Inc. | Methods of identifying somatic mutational signatures for early cancer detection |
EP4036578A1 (en) | 2016-11-11 | 2022-08-03 | Isoplexis Corporation | Compositions and methods for the simultaneous genomic, transcriptomic and proteomic analysis of single cells |
WO2018098372A1 (en) | 2016-11-22 | 2018-05-31 | IsoPlexis Corporation | Systems, devices and methods for cell capture and methods of manufacture thereof |
US11268137B2 (en) | 2016-12-09 | 2022-03-08 | Boreal Genomics, Inc. | Linked ligation |
US20180163201A1 (en) | 2016-12-12 | 2018-06-14 | Grail, Inc. | Methods for tagging and amplifying rna template molecules for preparing sequencing libraries |
EP3554514A4 (en) | 2016-12-16 | 2020-08-05 | Twist Bioscience Corporation | Variant libraries of the immunological synapse and synthesis thereof |
EP3555290B1 (en) | 2016-12-19 | 2022-11-02 | Bio-Rad Laboratories, Inc. | Droplet tagging contiguity preserved tagmented dna |
US10982351B2 (en) | 2016-12-23 | 2021-04-20 | Grail, Inc. | Methods for high efficiency library preparation using double-stranded adapters |
EP3574424A1 (en) | 2017-01-24 | 2019-12-04 | Sequenom, Inc. | Methods and processes for assessment of genetic variations |
DK3354746T3 (en) | 2017-01-30 | 2019-09-02 | Gmi Gregor Mendel Inst Fuer Molekulare Pflanzenbiologie Gmbh | NEW SPIKE-IN NUCLEOTIDES FOR NORMALIZING SEQUENCE DATA |
US10240205B2 (en) | 2017-02-03 | 2019-03-26 | Population Bio, Inc. | Methods for assessing risk of developing a viral disease using a genetic test |
EP3586255A4 (en) | 2017-02-22 | 2021-03-31 | Twist Bioscience Corporation | Nucleic acid based data storage |
BR112019018272A2 (en) | 2017-03-02 | 2020-07-28 | Youhealth Oncotech, Limited | methylation markers to diagnose hepatocellular carcinoma and cancer |
WO2018170169A1 (en) | 2017-03-15 | 2018-09-20 | Twist Bioscience Corporation | Variant libraries of the immunological synapse and synthesis thereof |
WO2018183918A1 (en) | 2017-03-30 | 2018-10-04 | Grail, Inc. | Enhanced ligation in sequencing library preparation |
US11118222B2 (en) | 2017-03-31 | 2021-09-14 | Grail, Inc. | Higher target capture efficiency using probe extension |
WO2018183942A1 (en) | 2017-03-31 | 2018-10-04 | Grail, Inc. | Improved library preparation and use thereof for sequencing-based error correction and/or variant identification |
CA3059370C (en) | 2017-04-12 | 2022-05-10 | Karius, Inc. | Methods for concurrent analysis of dna and rna in mixed samples |
WO2018195091A1 (en) | 2017-04-18 | 2018-10-25 | Dovetail Genomics, Llc | Nucleic acid characteristics as guides for sequence assembly |
WO2018209165A1 (en) | 2017-05-12 | 2018-11-15 | Laboratory Corporation Of America Holdings | Systems and methods for biomarker identificaton |
JP7169993B2 (en) | 2017-05-12 | 2022-11-11 | ラボラトリー コーポレイション オブ アメリカ ホールディングス | Compositions and methods for detecting non-celiac-gluten sensitivity |
WO2018213803A1 (en) | 2017-05-19 | 2018-11-22 | Neon Therapeutics, Inc. | Immunogenic neoantigen identification |
WO2018218222A1 (en) | 2017-05-26 | 2018-11-29 | Goldfless Stephen Jacob | High-throughput polynucleotide library sequencing and transcriptome analysis |
WO2018227091A1 (en) | 2017-06-08 | 2018-12-13 | The Brigham And Women's Hospital, Inc. | Methods and compositions for identifying epitopes |
WO2018231864A1 (en) | 2017-06-12 | 2018-12-20 | Twist Bioscience Corporation | Methods for seamless nucleic acid assembly |
AU2018284227A1 (en) | 2017-06-12 | 2020-01-30 | Twist Bioscience Corporation | Methods for seamless nucleic acid assembly |
CN110770356A (en) | 2017-06-20 | 2020-02-07 | 生物辐射实验室股份有限公司 | MDA using bead oligonucleotides |
CA3068273A1 (en) | 2017-06-21 | 2018-12-27 | Bluedot Llc | Systems and methods for identification of nucleic acids in a sample |
WO2019002265A1 (en) | 2017-06-26 | 2019-01-03 | Universität Für Bodenkultur Wien | Novel biomarkers for detecting senescent cells |
EP3545106B1 (en) | 2017-08-01 | 2022-01-19 | Helitec Limited | Methods of enriching and determining target nucleotide sequences |
EP3679370A1 (en) | 2017-09-07 | 2020-07-15 | Juno Therapeutics, Inc. | Methods of identifying cellular attributes related to outcomes associated with cell therapy |
KR20200047706A (en) | 2017-09-11 | 2020-05-07 | 트위스트 바이오사이언스 코포레이션 | GPCR binding protein and method for synthesis thereof |
WO2019055819A1 (en) | 2017-09-14 | 2019-03-21 | Grail, Inc. | Methods for preparing a sequencing library from single-stranded dna |
WO2019055829A1 (en) | 2017-09-15 | 2019-03-21 | Nazarian Javad | Methods for detecting cancer biomarkers |
KR20200057024A (en) | 2017-09-20 | 2020-05-25 | 가던트 헬쓰, 인크. | Methods and systems for differentiating somatic and germline variants |
WO2019060716A1 (en) | 2017-09-25 | 2019-03-28 | Freenome Holdings, Inc. | Methods and systems for sample extraction |
US11851650B2 (en) | 2017-09-28 | 2023-12-26 | Grail, Llc | Enrichment of short nucleic acid fragments in sequencing library preparation |
JP7260553B2 (en) | 2017-10-06 | 2023-04-18 | ザ・ユニバーシティ・オブ・シカゴ | Screening of T lymphocytes against cancer-specific antigens |
US11099202B2 (en) | 2017-10-20 | 2021-08-24 | Tecan Genomics, Inc. | Reagent delivery system |
SG11202003574TA (en) | 2017-10-20 | 2020-05-28 | Twist Bioscience Corp | Heated nanowells for polynucleotide synthesis |
EP4180534A1 (en) | 2017-11-02 | 2023-05-17 | Bio-Rad Laboratories, Inc. | Transposase-based genomic analysis |
JP7038209B2 (en) | 2017-11-13 | 2022-03-17 | エフ.ホフマン-ラ ロシュ アーゲー | Equipment for sample analysis using epitaco electrophoresis |
US11414656B2 (en) | 2017-12-15 | 2022-08-16 | Grail, Inc. | Methods for enriching for duplex reads in sequencing and error correction |
WO2019126249A1 (en) | 2017-12-20 | 2019-06-27 | Laboratory Corporation Of America Holdings | Compositions and methods to detect head and neck cancer |
WO2019126803A1 (en) | 2017-12-22 | 2019-06-27 | Grail, Inc. | Error removal using improved library preparation methods |
CA3088911A1 (en) | 2018-01-04 | 2019-07-11 | Twist Bioscience Corporation | Dna-based digital information storage |
US11366303B2 (en) | 2018-01-30 | 2022-06-21 | Rebus Biosystems, Inc. | Method for detecting particles using structured illumination |
EP4324962A2 (en) | 2018-01-31 | 2024-02-21 | Bio-Rad Laboratories, Inc. | Methods and compositions for deconvoluting partition barcodes |
WO2019152543A1 (en) | 2018-01-31 | 2019-08-08 | Dovetail Genomics, Llc | Sample prep for dna linkage recovery |
US11512002B2 (en) | 2018-04-18 | 2022-11-29 | University Of Virginia Patent Foundation | Silica materials and methods of making thereof |
WO2019213619A1 (en) | 2018-05-04 | 2019-11-07 | Abbott Laboratories | Hbv diagnostic, prognostic, and therapeutic methods and products |
WO2019217899A2 (en) | 2018-05-11 | 2019-11-14 | Laboratory Corporation Of America Holdings | Compositions and methods to detect kidney fibrosis |
WO2019222706A1 (en) | 2018-05-18 | 2019-11-21 | Twist Bioscience Corporation | Polynucleotides, reagents, and methods for nucleic acid hybridization |
US11814750B2 (en) | 2018-05-31 | 2023-11-14 | Personalis, Inc. | Compositions, methods and systems for processing or analyzing multi-species nucleic acid samples |
US10801064B2 (en) | 2018-05-31 | 2020-10-13 | Personalis, Inc. | Compositions, methods and systems for processing or analyzing multi-species nucleic acid samples |
US20190385700A1 (en) | 2018-06-04 | 2019-12-19 | Guardant Health, Inc. | METHODS AND SYSTEMS FOR DETERMINING The CELLULAR ORIGIN OF CELL-FREE NUCLEIC ACIDS |
AU2019310041A1 (en) | 2018-07-23 | 2021-02-04 | Guardant Health, Inc. | Methods and systems for adjusting tumor mutational burden by tumor fraction and coverage |
SI3625368T1 (en) | 2018-08-08 | 2023-04-28 | Pml Screening, Llc | Methods for assessing the risk of developing progressive multifocal leukoencephalopathy caused by john cunningham virus by genetic testing |
EP3841202B1 (en) | 2018-08-20 | 2023-10-04 | Bio-Rad Laboratories, Inc. | Nucleotide sequence generation by barcode bead-colocalization in partitions |
AU2019331907A1 (en) | 2018-08-30 | 2021-04-08 | Guardant Health, Inc. | Methods and systems for detecting contamination between samples |
CA3109539A1 (en) | 2018-08-31 | 2020-03-05 | Guardant Health, Inc. | Microsatellite instability detection in cell-free dna |
WO2020096691A2 (en) | 2018-09-04 | 2020-05-14 | Guardant Health, Inc. | Methods and systems for detecting allelic imbalance in cell-free nucleic acid samples |
DK3814533T3 (en) | 2018-09-20 | 2021-11-15 | Tamirna Gmbh | Micro-RNA signatures to predict liver dysfunction |
WO2020074742A1 (en) | 2018-10-12 | 2020-04-16 | F. Hoffmann-La Roche Ag | Detection methods for epitachophoresis workflow automation |
EP3874060A1 (en) | 2018-10-31 | 2021-09-08 | Guardant Health, Inc. | Methods, compositions and systems for calibrating epigenetic partitioning assays |
US10876148B2 (en) | 2018-11-14 | 2020-12-29 | Element Biosciences, Inc. | De novo surface preparation and uses thereof |
US10768173B1 (en) | 2019-09-06 | 2020-09-08 | Element Biosciences, Inc. | Multivalent binding composition for nucleic acid analysis |
US10704094B1 (en) | 2018-11-14 | 2020-07-07 | Element Biosciences, Inc. | Multipart reagents having increased avidity for polymerase binding |
US11680261B2 (en) | 2018-11-15 | 2023-06-20 | Grail, Inc. | Needle-based devices and methods for in vivo diagnostics of disease conditions |
CA3121805A1 (en) | 2018-12-07 | 2020-06-11 | Octant, Inc. | Systems for protein-protein interaction screening |
JP2022512328A (en) | 2018-12-07 | 2022-02-03 | エレメント バイオサイエンシーズ,インク. | Flow cell device and its use |
CN113286883A (en) | 2018-12-18 | 2021-08-20 | 格里尔公司 | Methods for detecting disease using RNA analysis |
CN113454218A (en) | 2018-12-20 | 2021-09-28 | 夸登特健康公司 | Methods, compositions, and systems for improved recovery of nucleic acid molecules |
WO2020141464A1 (en) | 2019-01-03 | 2020-07-09 | Boreal Genomics, Inc. | Linked target capture |
US20220081714A1 (en) | 2019-01-04 | 2022-03-17 | Northwestern University | Storing temporal data into dna |
WO2020160414A1 (en) | 2019-01-31 | 2020-08-06 | Guardant Health, Inc. | Compositions and methods for isolating cell-free dna |
US11492727B2 (en) | 2019-02-26 | 2022-11-08 | Twist Bioscience Corporation | Variant nucleic acid libraries for GLP1 receptor |
WO2020176680A1 (en) | 2019-02-26 | 2020-09-03 | Twist Bioscience Corporation | Variant nucleic acid libraries for antibody optimization |
WO2020176659A1 (en) | 2019-02-27 | 2020-09-03 | Guardant Health, Inc. | Methods and systems for determining the cellular origin of cell-free dna |
US11578373B2 (en) | 2019-03-26 | 2023-02-14 | Dermtech, Inc. | Gene classifiers and uses thereof in skin cancers |
US20220325268A1 (en) | 2019-05-14 | 2022-10-13 | Roche Sequencing Solutions, Inc | Devices and methods for sample analysis |
CA3140902A1 (en) | 2019-05-28 | 2020-12-03 | Octant, Inc. | Transcriptional relay system |
EP3976822A1 (en) | 2019-05-31 | 2022-04-06 | Guardant Health, Inc. | Methods and systems for improving patient monitoring after surgery |
CN114729342A (en) | 2019-06-21 | 2022-07-08 | 特韦斯特生物科学公司 | Barcode-based nucleic acid sequence assembly |
JP7194311B2 (en) | 2019-07-10 | 2022-12-21 | ウルティマ ジェノミクス, インコーポレイテッド | RNA sequencing method |
US11287422B2 (en) | 2019-09-23 | 2022-03-29 | Element Biosciences, Inc. | Multivalent binding composition for nucleic acid analysis |
JP2022552194A (en) | 2019-10-10 | 2022-12-15 | 1859,インク. | Methods and systems for microfluidic screening |
DK3812472T3 (en) | 2019-10-21 | 2023-02-20 | Univ Freiburg Albert Ludwigs | TRULY UNBIASED IN VITRO ASSAYS FOR PROFILING THE OFF-TARGET ACTIVITY OF ONE OR MORE TARGET-SPECIFIC PROGRAMMABLE NUCLEASES IN CELLS (ABNOBA-SEQ) |
CN114746560A (en) | 2019-11-26 | 2022-07-12 | 夸登特健康公司 | Methods, compositions, and systems for improved binding of methylated polynucleotides |
WO2021152586A1 (en) | 2020-01-30 | 2021-08-05 | Yeda Research And Development Co. Ltd. | Methods of analyzing microbiome, immunoglobulin profile and physiological state |
WO2021214766A1 (en) | 2020-04-21 | 2021-10-28 | Yeda Research And Development Co. Ltd. | Methods of diagnosing viral infections and vaccines thereto |
WO2021222828A1 (en) | 2020-04-30 | 2021-11-04 | Guardant Health, Inc. | Methods for sequence determination using partitioned nucleic acids |
WO2021224677A1 (en) | 2020-05-05 | 2021-11-11 | Akershus Universitetssykehus Hf | Compositions and methods for characterizing bowel cancer |
JP2023526252A (en) | 2020-05-14 | 2023-06-21 | ガーダント ヘルス, インコーポレイテッド | Detection of homologous recombination repair defects |
WO2023282916A1 (en) | 2021-07-09 | 2023-01-12 | Guardant Health, Inc. | Methods of detecting genomic rearrangements using cell free nucleic acids |
EP4189111A1 (en) | 2020-07-30 | 2023-06-07 | Guardant Health, Inc. | Methods for isolating cell-free dna |
EP4205126A1 (en) | 2020-08-25 | 2023-07-05 | Guardant Health, Inc. | Methods and systems for predicting an origin of a variant |
US20220154285A1 (en) | 2020-09-30 | 2022-05-19 | Guardant Health, Inc. | Analysis of methylated dna comprising methylation-sensitive or methylation-dependent restrictions |
EP4267757A1 (en) | 2020-12-23 | 2023-11-01 | Guardant Health, Inc. | Methods and systems for analyzing methylated polynucleotides |
KR20230156364A (en) | 2021-03-05 | 2023-11-14 | 가던트 헬쓰, 인크. | Methods and related aspects for analyzing molecular reactions |
US20220344004A1 (en) | 2021-03-09 | 2022-10-27 | Guardant Health, Inc. | Detecting the presence of a tumor based on off-target polynucleotide sequencing data |
EP4308723A1 (en) | 2021-03-15 | 2024-01-24 | F. Hoffmann-La Roche AG | Targeted next-generation sequencing via anchored primer extension |
WO2022204321A1 (en) | 2021-03-24 | 2022-09-29 | Ambry Genetics Corporation | Conservative concurrent evaluation of dna modifications |
WO2022208171A1 (en) | 2021-03-31 | 2022-10-06 | UCL Business Ltd. | Methods for analyte detection |
WO2023004344A1 (en) | 2021-07-20 | 2023-01-26 | Regeneron Pharmaceuticals, Inc. | Butyrophilin-like 2 for treating inflammatory disorders |
WO2023035003A1 (en) | 2021-09-03 | 2023-03-09 | Elegen Corp. | Multi-way bead-sorting devices, systems, and methods of use thereof using pressure sources |
EP4253550A1 (en) | 2022-04-01 | 2023-10-04 | GenCC GmbH 6 Co. KG | Method for the manufacture of a viral system, a vector system or any transport system for cancer-specific crispr complexes |
US20230360725A1 (en) | 2022-05-09 | 2023-11-09 | Guardant Health, Inc. | Detecting degradation based on strand bias |
WO2023218408A1 (en) | 2022-05-11 | 2023-11-16 | Freya Biosciences Aps | Methods of identifying strains associated with the human female genitourinary tract |
Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1993021340A1 (en) * | 1992-04-22 | 1993-10-28 | Medical Research Council | Dna sequencing method |
US5502773A (en) * | 1991-09-20 | 1996-03-26 | Vanderbilt University | Method and apparatus for automated processing of DNA sequence data |
WO1996027025A1 (en) * | 1995-02-27 | 1996-09-06 | Ely Michael Rabani | Device, compounds, algorithms, and methods of molecular characterization and manipulation with molecular parallelism |
US20020025529A1 (en) * | 1999-06-28 | 2002-02-28 | Stephen Quake | Methods and apparatus for analyzing polynucleotide sequences |
US20020164629A1 (en) * | 2001-03-12 | 2002-11-07 | California Institute Of Technology | Methods and apparatus for analyzing polynucleotide sequences by asynchronous base extension |
US20050170367A1 (en) * | 2003-06-10 | 2005-08-04 | Quake Stephen R. | Fluorescently labeled nucleoside triphosphates and analogs thereof for sequencing nucleic acids |
US7169560B2 (en) * | 2003-11-12 | 2007-01-30 | Helicos Biosciences Corporation | Short cycle methods for sequencing polynucleotides |
US20090005259A1 (en) * | 2003-02-26 | 2009-01-01 | Complete Genomics, Inc. | Random array DNA analysis by hybridization |
Family Cites Families (475)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3996345A (en) | 1974-08-12 | 1976-12-07 | Syva Company | Fluorescence quenching with immunological pairs in immunoassays |
JPS5941169B2 (en) | 1975-12-25 | 1984-10-05 | シチズン時計株式会社 | Elastomer |
US4153855A (en) | 1977-12-16 | 1979-05-08 | The United States Of America As Represented By The Secretary Of The Army | Method of making a plate having a pattern of microchannels |
US4351760A (en) | 1979-09-07 | 1982-09-28 | Syva Company | Novel alkyl substituted fluorescent compounds and polyamino acid conjugates |
US4344064A (en) | 1979-12-06 | 1982-08-10 | Western Electric Co., Inc. | Article carrying a distinctive mark |
US4711955A (en) | 1981-04-17 | 1987-12-08 | Yale University | Modified nucleotides and methods of preparing and using same |
US5260433A (en) | 1982-06-23 | 1993-11-09 | Enzo Diagnostics, Inc. | Saccharide specific binding system labeled nucleotides |
US4707237A (en) | 1982-09-09 | 1987-11-17 | Ciba Corning Diagnostics Corp. | System for identification of cells by electrophoresis |
US5198540A (en) | 1982-10-28 | 1993-03-30 | Hubert Koster | Process for the preparation of oligonucleotides in solution |
US4994373A (en) | 1983-01-27 | 1991-02-19 | Enzo Biochem, Inc. | Method and structures employing chemically-labelled polynucleotide probes |
USRE34069E (en) | 1983-08-18 | 1992-09-15 | Biosyntech Gmbh | Process for the preparation of oligonucleotides |
DE3329892A1 (en) | 1983-08-18 | 1985-03-07 | Köster, Hubert, Prof. Dr., 2000 Hamburg | METHOD FOR PRODUCING OLIGONUCLEOTIDES |
US5821058A (en) | 1984-01-16 | 1998-10-13 | California Institute Of Technology | Automated DNA sequencing technique |
US4581624A (en) | 1984-03-01 | 1986-04-08 | Allied Corporation | Microminiature semiconductor valve |
US5360523A (en) | 1984-03-29 | 1994-11-01 | Li-Cor, Inc. | DNA sequencing |
US4729947A (en) | 1984-03-29 | 1988-03-08 | The Board Of Regents Of The University Of Nebraska | DNA sequencing |
US5258506A (en) | 1984-10-16 | 1993-11-02 | Chiron Corporation | Photolabile reagents for incorporation into oligonucleotide chains |
US6060237A (en) | 1985-02-26 | 2000-05-09 | Biostar, Inc. | Devices and methods for optical detection of nucleic acid hybridization |
US4683195A (en) | 1986-01-30 | 1987-07-28 | Cetus Corporation | Process for amplifying, detecting, and/or-cloning nucleic acid sequences |
US4683202A (en) | 1985-03-28 | 1987-07-28 | Cetus Corporation | Process for amplifying nucleic acid sequences |
US4865968A (en) | 1985-04-01 | 1989-09-12 | The Salk Institute For Biological Studies | DNA sequencing |
US4739044A (en) | 1985-06-13 | 1988-04-19 | Amgen | Method for derivitization of polynucleotides |
US4863849A (en) | 1985-07-18 | 1989-09-05 | New York Medical College | Automatable process for sequencing nucleotide |
US4757141A (en) | 1985-08-26 | 1988-07-12 | Applied Biosystems, Incorporated | Amino-derivatized phosphite and phosphate linking agents, phosphoramidite precursors, and useful conjugates thereof |
US4855225A (en) | 1986-02-07 | 1989-08-08 | Applied Biosystems, Inc. | Method of detecting electrophoretically separated oligonucleotides |
US5242797A (en) | 1986-03-21 | 1993-09-07 | Myron J. Block | Nucleic acid assay method |
US4811218A (en) | 1986-06-02 | 1989-03-07 | Applied Biosystems, Inc. | Real time scanning electrophoresis apparatus for DNA sequencing |
CA1340806C (en) | 1986-07-02 | 1999-11-02 | James Merrill Prober | Method, system and reagents for dna sequencing |
US5242796A (en) | 1986-07-02 | 1993-09-07 | E. I. Du Pont De Nemours And Company | Method, system and reagents for DNA sequencing |
US4889818A (en) | 1986-08-22 | 1989-12-26 | Cetus Corporation | Purified thermostable enzyme |
US4994372A (en) | 1987-01-14 | 1991-02-19 | President And Fellows Of Harvard College | DNA sequencing |
US5001060A (en) | 1987-02-06 | 1991-03-19 | Lubrizol Enterprises, Inc. | Plant anaerobic regulatory element |
US5525464A (en) | 1987-04-01 | 1996-06-11 | Hyseq, Inc. | Method of sequencing by hybridization of oligonucleotide probes |
US5202231A (en) | 1987-04-01 | 1993-04-13 | Drmanac Radoje T | Method of sequencing of genomes by hybridization of oligonucleotide probes |
US6270961B1 (en) | 1987-04-01 | 2001-08-07 | Hyseq, Inc. | Methods and apparatus for DNA sequencing and DNA identification |
US4994368A (en) | 1987-07-23 | 1991-02-19 | Syntex (U.S.A.) Inc. | Amplification method for polynucleotide assays |
US4942124A (en) | 1987-08-11 | 1990-07-17 | President And Fellows Of Harvard College | Multiplex sequencing |
US4793705A (en) | 1987-10-07 | 1988-12-27 | The United States Of America As Represented By The United States Department Of Energy | Single molecule tracking |
US4962037A (en) | 1987-10-07 | 1990-10-09 | United States Of America | Method for rapid base sequencing in DNA and RNA |
AU622426B2 (en) | 1987-12-11 | 1992-04-09 | Abbott Laboratories | Assay using template-dependent nucleic acid probe reorganization |
US4971903A (en) | 1988-03-25 | 1990-11-20 | Edward Hyman | Pyrophosphate-based method and apparatus for sequencing nucleic acids |
US4962020A (en) | 1988-07-12 | 1990-10-09 | President And Fellows Of Harvard College | DNA sequencing |
CH679555A5 (en) | 1989-04-11 | 1992-03-13 | Westonbridge Int Ltd | |
GB8910880D0 (en) | 1989-05-11 | 1989-06-28 | Amersham Int Plc | Sequencing method |
US4979824A (en) | 1989-05-26 | 1990-12-25 | Board Of Trustees Of The Leland Stanford Junior University | High sensitivity fluorescent single particle and single molecule detection apparatus and method |
US5800992A (en) | 1989-06-07 | 1998-09-01 | Fodor; Stephen P.A. | Method of detecting nucleic acids |
US6346413B1 (en) | 1989-06-07 | 2002-02-12 | Affymetrix, Inc. | Polymer arrays |
US5424186A (en) | 1989-06-07 | 1995-06-13 | Affymax Technologies N.V. | Very large scale immobilized polymer synthesis |
US6551784B2 (en) | 1989-06-07 | 2003-04-22 | Affymetrix Inc | Method of comparing nucleic acid sequences |
US5547839A (en) | 1989-06-07 | 1996-08-20 | Affymax Technologies N.V. | Sequencing of surface immobilized polymers utilizing microflourescence detection |
US5744101A (en) | 1989-06-07 | 1998-04-28 | Affymax Technologies N.V. | Photolabile nucleoside protecting groups |
US6416952B1 (en) | 1989-06-07 | 2002-07-09 | Affymetrix, Inc. | Photolithographic and other means for manufacturing arrays |
US5143854A (en) | 1989-06-07 | 1992-09-01 | Affymax Technologies N.V. | Large scale photolithographic solid phase synthesis of polypeptides and receptor binding screening thereof |
US5112736A (en) | 1989-06-14 | 1992-05-12 | University Of Utah | Dna sequencing using fluorescence background electroblotting membrane |
DE69011631T2 (en) | 1989-06-14 | 1995-03-23 | Westonbridge Int Ltd | MICRO PUMP. |
US5108892A (en) | 1989-08-03 | 1992-04-28 | Promega Corporation | Method of using a taq dna polymerase without 5'-3'-exonuclease activity |
US5096554A (en) | 1989-08-07 | 1992-03-17 | Applied Biosystems, Inc. | Nucleic acid fractionation by counter-migration capillary electrophoresis |
FR2650840B1 (en) | 1989-08-11 | 1991-11-29 | Bertin & Cie | RAPID DETECTION AND / OR IDENTIFICATION OF A SINGLE BASED ON A NUCLEIC ACID SEQUENCE, AND ITS APPLICATIONS |
US5302509A (en) | 1989-08-14 | 1994-04-12 | Beckman Instruments, Inc. | Method for sequencing polynucleotides |
AU649407B2 (en) | 1989-09-21 | 1994-05-26 | Infigen, Inc. | Enhanced gene expression in response to lactation signals |
WO1991006678A1 (en) | 1989-10-26 | 1991-05-16 | Sri International | Dna sequencing |
CA2028261C (en) | 1989-10-28 | 1995-01-17 | Won Suck Yang | Non-invasive method and apparatus for measuring blood glucose concentration |
US5171132A (en) | 1989-12-27 | 1992-12-15 | Seiko Epson Corporation | Two-valve thin plate micropump |
US5091652A (en) | 1990-01-12 | 1992-02-25 | The Regents Of The University Of California | Laser excited confocal microscope fluorescence scanner and method |
WO1991011533A1 (en) | 1990-01-26 | 1991-08-08 | E.I. Du Pont De Nemours And Company | Method for isolating primer extension products from template-directed dna polymerase reactions |
DE4006152A1 (en) | 1990-02-27 | 1991-08-29 | Fraunhofer Ges Forschung | MICROMINIATURIZED PUMP |
US5096388A (en) | 1990-03-22 | 1992-03-17 | The Charles Stark Draper Laboratory, Inc. | Microfabricated pump |
SE470347B (en) | 1990-05-10 | 1994-01-31 | Pharmacia Lkb Biotech | Microstructure for fluid flow systems and process for manufacturing such a system |
EP0465229B1 (en) | 1990-07-02 | 1994-12-28 | Seiko Epson Corporation | Micropump and process for manufacturing a micropump |
EP0834576B1 (en) | 1990-12-06 | 2002-01-16 | Affymetrix, Inc. (a Delaware Corporation) | Detection of nucleic acid sequences |
ES2155822T3 (en) | 1990-12-06 | 2001-06-01 | Affymetrix Inc | COMPOUNDS AND ITS USE IN A BINARY SYNTHESIS STRATEGY. |
US5198340A (en) * | 1991-01-17 | 1993-03-30 | Genentech, Inc. | Assay for free igf-i, igf-ii, and gh levels in body fluids |
US5529679A (en) | 1992-02-28 | 1996-06-25 | Hitachi, Ltd. | DNA detector and DNA detection method |
US5888819A (en) | 1991-03-05 | 1999-03-30 | Molecular Tool, Inc. | Method for determining nucleotide identity through primer extension |
US6004744A (en) | 1991-03-05 | 1999-12-21 | Molecular Tool, Inc. | Method for determining nucleotide identity through extension of immobilized primer |
US5518900A (en) | 1993-01-15 | 1996-05-21 | Molecular Tool, Inc. | Method for generating single-stranded DNA molecules |
US5762876A (en) | 1991-03-05 | 1998-06-09 | Molecular Tool, Inc. | Automatic genotype determination |
US5167784A (en) | 1991-09-04 | 1992-12-01 | Xerox Corporation | Sequencing large nucleic acid fragments |
WO1993005183A1 (en) | 1991-09-09 | 1993-03-18 | Baylor College Of Medicine | Method and device for rapid dna or rna sequencing determination by a base addition sequencing scheme |
DE4143343C2 (en) | 1991-09-11 | 1994-09-22 | Fraunhofer Ges Forschung | Microminiaturized, electrostatically operated micromembrane pump |
US5265327A (en) | 1991-09-13 | 1993-11-30 | Faris Sadeg M | Microchannel plate technology |
AU669489B2 (en) | 1991-09-18 | 1996-06-13 | Affymax Technologies N.V. | Method of synthesizing diverse collections of oligomers |
US5405747A (en) | 1991-09-25 | 1995-04-11 | The Regents Of The University Of California Office Of Technology Transfer | Method for rapid base sequencing in DNA and RNA with two base labeling |
US5756285A (en) | 1991-09-27 | 1998-05-26 | Amersham Life Science, Inc. | DNA cycle sequencing |
US5674679A (en) | 1991-09-27 | 1997-10-07 | Amersham Life Science, Inc. | DNA cycle sequencing |
US5632957A (en) | 1993-11-01 | 1997-05-27 | Nanogen | Molecular biological diagnostic systems including electrodes |
US6051380A (en) | 1993-11-01 | 2000-04-18 | Nanogen, Inc. | Methods and procedures for molecular biological analysis and diagnostics |
JP3509859B2 (en) | 1991-11-07 | 2004-03-22 | ナノトロニクス,インコーポレイテッド | Hybridization of chromophore and fluorophore conjugated polynucleotides to create donor-donor energy transfer systems |
CA2125144A1 (en) | 1991-12-23 | 1993-07-08 | Chiron Diagnostics Corporation | Htlv-1 probes for use in solution phase sandwich hybridization assays |
US6555349B1 (en) | 1993-01-22 | 2003-04-29 | Cornell Research Foundation, Inc. | Methods for amplifying and sequencing nucleic acid molecules using a three component polymerase |
US5209834A (en) | 1992-03-09 | 1993-05-11 | The United States Of America As Represented By The United States Department Of Energy | Ordered transport and identification of particles |
US5304487A (en) | 1992-05-01 | 1994-04-19 | Trustees Of The University Of Pennsylvania | Fluid handling in mesoscale analytical devices |
DE4220077A1 (en) | 1992-06-19 | 1993-12-23 | Bosch Gmbh Robert | Micro-pump for delivery of gases - uses working chamber warmed by heating element and controlled by silicon wafer valves. |
JPH0622798A (en) | 1992-07-07 | 1994-02-01 | Hitachi Ltd | Method for determining base sequence |
US5284568A (en) | 1992-07-17 | 1994-02-08 | E. I. Du Pont De Nemours And Company | Disposable cartridge for ion selective electrode sensors |
US5306403A (en) | 1992-08-24 | 1994-04-26 | Martin Marietta Energy Systems, Inc. | Raman-based system for DNA sequencing-mapping and other separations |
US5403709A (en) | 1992-10-06 | 1995-04-04 | Hybridon, Inc. | Method for sequencing synthetic oligonucleotides containing non-phosphodiester internucleotide linkages |
US6436635B1 (en) | 1992-11-06 | 2002-08-20 | Boston University | Solid phase sequencing of double-stranded nucleic acids |
US5605798A (en) | 1993-01-07 | 1997-02-25 | Sequenom, Inc. | DNA diagnostic based on mass spectrometry |
AU671820B2 (en) | 1993-02-10 | 1996-09-12 | Promega Corporation | Non-radioactive DNA sequencing |
US5436149A (en) | 1993-02-19 | 1995-07-25 | Barnes; Wayne M. | Thermostable DNA polymerase with enhanced thermostability and enhanced length and efficiency of primer extension |
EP0705271B1 (en) | 1993-06-25 | 2002-11-13 | Affymetrix, Inc. (a Delaware Corporation) | Hybridization and sequencing of nucleic acids |
US5837832A (en) | 1993-06-25 | 1998-11-17 | Affymetrix, Inc. | Arrays of nucleic acid probes on biological chips |
US5547859A (en) | 1993-08-02 | 1996-08-20 | Goodman; Myron F. | Chain-terminating nucleotides for DNA sequencing methods |
DE4326466A1 (en) | 1993-08-06 | 1995-02-09 | Boehringer Mannheim Gmbh | Infrared dye-labeled nucleotides and their use in nucleic acid detection |
US6376178B1 (en) | 1993-09-03 | 2002-04-23 | Duke University | Method of nucleic acid sequencing |
US5659171A (en) | 1993-09-22 | 1997-08-19 | Northrop Grumman Corporation | Micro-miniature diaphragm pump for the low pressure pumping of gases |
US6401267B1 (en) | 1993-09-27 | 2002-06-11 | Radoje Drmanac | Methods and compositions for efficient nucleic acid sequencing |
US5470710A (en) | 1993-10-22 | 1995-11-28 | University Of Utah | Automated hybridization/imaging device for fluorescent multiplex DNA sequencing |
US6156501A (en) | 1993-10-26 | 2000-12-05 | Affymetrix, Inc. | Arrays of modified nucleic acid probes and methods of use |
US6309601B1 (en) | 1993-11-01 | 2001-10-30 | Nanogen, Inc. | Scanning optical detection system |
WO1995012608A1 (en) | 1993-11-02 | 1995-05-11 | Affymax Technologies N.V. | Synthesizing and screening molecular diversity |
US5610287A (en) | 1993-12-06 | 1997-03-11 | Molecular Tool, Inc. | Method for immobilizing nucleic acid molecules |
CH689836A5 (en) | 1994-01-14 | 1999-12-15 | Westonbridge Int Ltd | Micropump. |
US6028190A (en) | 1994-02-01 | 2000-02-22 | The Regents Of The University Of California | Probes labeled with energy transfer coupled dyes |
US5654419A (en) | 1994-02-01 | 1997-08-05 | The Regents Of The University Of California | Fluorescent labels and their use in separations |
EP0754240B1 (en) | 1994-02-07 | 2003-08-20 | Beckman Coulter, Inc. | Ligase/polymerase-mediated genetic bit analysis of single nucleotide polymorphisms and its use in genetic analysis |
US5578832A (en) | 1994-09-02 | 1996-11-26 | Affymetrix, Inc. | Method and apparatus for imaging a sample on a device |
US5631734A (en) | 1994-02-10 | 1997-05-20 | Affymetrix, Inc. | Method and apparatus for detection of fluorescently labeled materials |
JP3448090B2 (en) | 1994-02-16 | 2003-09-16 | 浜松ホトニクス株式会社 | Energy transfer detection method and apparatus |
AU1854895A (en) | 1994-03-08 | 1995-09-25 | Amersham International Plc | Modifying nucleotide analogues |
US5552278A (en) | 1994-04-04 | 1996-09-03 | Spectragen, Inc. | DNA sequencing by stepwise ligation and cleavage |
US20020168642A1 (en) | 1994-06-06 | 2002-11-14 | Andrzej Drukier | Sequencing duplex DNA by mass spectroscopy |
US6287850B1 (en) | 1995-06-07 | 2001-09-11 | Affymetrix, Inc. | Bioarray chip reaction apparatus and its manufacture |
US6071394A (en) | 1996-09-06 | 2000-06-06 | Nanogen, Inc. | Channel-less separation of bioparticles on a bioelectronic chip by dielectrophoresis |
US6379897B1 (en) | 2000-11-09 | 2002-04-30 | Nanogen, Inc. | Methods for gene expression monitoring on electronic microarrays |
US5834189A (en) | 1994-07-08 | 1998-11-10 | Visible Genetics Inc. | Method for evaluation of polymorphic genetic sequences, and the use thereof in identification of HLA types |
US5853979A (en) | 1995-06-30 | 1998-12-29 | Visible Genetics Inc. | Method and system for DNA sequence determination and mutation detection with reference to a standard |
US6001229A (en) | 1994-08-01 | 1999-12-14 | Lockheed Martin Energy Systems, Inc. | Apparatus and method for performing microfluidic manipulations for chemical analysis |
US5808045A (en) | 1994-09-02 | 1998-09-15 | Andrew C. Hiatt | Compositions for enzyme catalyzed template-independent creation of phosphodiester bonds using protected nucleotides |
US5872244A (en) | 1994-09-02 | 1999-02-16 | Andrew C. Hiatt | 3' protected nucleotides for enzyme catalyzed template-independent creation of phosphodiester bonds |
US6214987B1 (en) * | 1994-09-02 | 2001-04-10 | Andrew C. Hiatt | Compositions for enzyme catalyzed template-independent formation of phosphodiester bonds using protected nucleotides |
US5763594A (en) | 1994-09-02 | 1998-06-09 | Andrew C. Hiatt | 3' protected nucleotides for enzyme catalyzed template-independent creation of phosphodiester bonds |
DE4433894A1 (en) | 1994-09-22 | 1996-03-28 | Fraunhofer Ges Forschung | Method and device for controlling a micropump |
US6015668A (en) | 1994-09-30 | 2000-01-18 | Life Technologies, Inc. | Cloned DNA polymerases from thermotoga and mutants thereof |
US5912155A (en) | 1994-09-30 | 1999-06-15 | Life Technologies, Inc. | Cloned DNA polymerases from Thermotoga neapolitana |
DE69531430T2 (en) | 1994-10-07 | 2004-07-01 | Bayer Corp. | relief valve |
US5695934A (en) | 1994-10-13 | 1997-12-09 | Lynx Therapeutics, Inc. | Massively parallel sequencing of sorted polynucleotides |
US5604097A (en) | 1994-10-13 | 1997-02-18 | Spectragen, Inc. | Methods for sorting polynucleotides using oligonucleotide tags |
US6654505B2 (en) | 1994-10-13 | 2003-11-25 | Lynx Therapeutics, Inc. | System and apparatus for sequential processing of analytes |
US5795716A (en) | 1994-10-21 | 1998-08-18 | Chee; Mark S. | Computer-aided visualization and analysis system for sequence evaluation |
US5707506A (en) | 1994-10-28 | 1998-01-13 | Battelle Memorial Institute | Channel plate for DNA sequencing |
US5514256A (en) | 1994-10-28 | 1996-05-07 | Battelle Memorial Institute | Apparatus for improved DNA sequencing |
FR2726286B1 (en) | 1994-10-28 | 1997-01-17 | Genset Sa | SOLID PHASE NUCLEIC ACID AMPLIFICATION PROCESS AND REAGENT KIT USEFUL FOR CARRYING OUT SAID PROCESS |
US5846396A (en) | 1994-11-10 | 1998-12-08 | Sarnoff Corporation | Liquid distribution system |
US5603351A (en) | 1995-06-07 | 1997-02-18 | David Sarnoff Research Center, Inc. | Method and system for inhibiting cross-contamination in fluids of combinatorial chemistry device |
US5556790A (en) | 1994-12-05 | 1996-09-17 | Pettit; John W. | Method for Automated DNA sequencing |
US5710628A (en) | 1994-12-12 | 1998-01-20 | Visible Genetics Inc. | Automated electrophoresis and fluorescence detection apparatus and method |
US5786142A (en) | 1995-05-30 | 1998-07-28 | Visible Genetics Inc. | Electrophoresis and fluorescence detection method |
SE9500342D0 (en) | 1995-01-31 | 1995-01-31 | Marek Kwiatkowski | Novel chain terminators, the use thereof for nucleic acid sequencing and synthesis and a method of their preparation |
US5599695A (en) | 1995-02-27 | 1997-02-04 | Affymetrix, Inc. | Printing molecular library arrays using deprotection agents solely in the vapor phase |
US5876187A (en) | 1995-03-09 | 1999-03-02 | University Of Washington | Micropumps with fixed valves |
US5795782A (en) | 1995-03-17 | 1998-08-18 | President & Fellows Of Harvard College | Characterization of individual polymer molecules based on monomer-interface interactions |
US6362002B1 (en) | 1995-03-17 | 2002-03-26 | President And Fellows Of Harvard College | Characterization of individual polymer molecules based on monomer-interface interactions |
US5994058A (en) | 1995-03-20 | 1999-11-30 | Genome International Corporation | Method for contiguous genome sequencing |
US5750341A (en) | 1995-04-17 | 1998-05-12 | Lynx Therapeutics, Inc. | DNA sequencing by parallel oligonucleotide extensions |
US5675155A (en) | 1995-04-26 | 1997-10-07 | Beckman Instruments, Inc. | Multicapillary fluorescent detection system |
US5830655A (en) | 1995-05-22 | 1998-11-03 | Sri International | Oligonucleotide sizing using cleavable primers |
CA2222744C (en) | 1995-05-31 | 2008-03-25 | Amersham Life Science, Inc. | Thermostable dna polymerases |
US6077664A (en) | 1995-06-07 | 2000-06-20 | Promega Corporation | Thermophilic DNA polymerases from Thermotoga neapolitana |
US5861287A (en) | 1995-06-23 | 1999-01-19 | Baylor College Of Medicine | Alternative dye-labeled primers for automated DNA sequencing |
CN1106937C (en) | 1995-06-26 | 2003-04-30 | 美国3M公司 | Multilayer polymer film with additional coatings or layers |
US5856174A (en) | 1995-06-29 | 1999-01-05 | Affymetrix, Inc. | Integrated nucleic acid diagnostic device |
US5981186A (en) | 1995-06-30 | 1999-11-09 | Visible Genetics, Inc. | Method and apparatus for DNA-sequencing using reduced number of sequencing mixtures |
US5916747A (en) | 1995-06-30 | 1999-06-29 | Visible Genetics Inc. | Method and apparatus for alignment of signals for use in DNA based-calling |
US5807679A (en) | 1995-07-07 | 1998-09-15 | Myriad Genetics, Inc. | Island hopping--a method to sequence rapidly very large fragments of DNA |
US5968740A (en) | 1995-07-24 | 1999-10-19 | Affymetrix, Inc. | Method of Identifying a Base in a Nucleic Acid |
DE19527155C2 (en) | 1995-07-25 | 1998-12-10 | Deutsches Krebsforsch | Method of sequencing by oligomer hybridization |
US5948614A (en) | 1995-09-08 | 1999-09-07 | Life Technologies, Inc. | Cloned DNA polymerases from thermotoga maritima and mutants thereof |
US5733729A (en) | 1995-09-14 | 1998-03-31 | Affymetrix, Inc. | Computer-aided probability base calling for arrays of nucleic acid probes on chips |
US6130098A (en) | 1995-09-15 | 2000-10-10 | The Regents Of The University Of Michigan | Moving microdroplets |
US5843655A (en) | 1995-09-18 | 1998-12-01 | Affymetrix, Inc. | Methods for testing oligonucleotide arrays |
US6132580A (en) | 1995-09-28 | 2000-10-17 | The Regents Of The University Of California | Miniature reaction chamber and devices incorporating same |
US6165765A (en) | 1995-10-18 | 2000-12-26 | Shanghai Institute Of Biochemistry, Chinese Academy Of Sciences | DNA polymerase having ability to reduce innate selective discrimination against fluorescent dye-labeled dideoxynucleotides |
SE9504099D0 (en) | 1995-11-16 | 1995-11-16 | Pharmacia Biotech Ab | A method of sequencing |
US5776767A (en) | 1995-12-12 | 1998-07-07 | Visible Genetics Inc. | Virtual DNA sequencer |
US5705018A (en) | 1995-12-13 | 1998-01-06 | Hartley; Frank T. | Micromachined peristaltic pump |
JP4073038B2 (en) | 1995-12-15 | 2008-04-09 | ジーイー・ヘルスケア・バイオサイエンス・コーポレイション | Thermostable DNA polymerase from Thermoanaerobacter thermohydrosulfurica and its mutant enzyme from which exonuclease activity has been removed |
US6147205A (en) | 1995-12-15 | 2000-11-14 | Affymetrix, Inc. | Photocleavable protecting groups and methods for their use |
WO1997022719A1 (en) | 1995-12-18 | 1997-06-26 | Washington University | Method for nucleic acid analysis using fluorescence resonance energy transfer |
KR100207410B1 (en) | 1995-12-19 | 1999-07-15 | 전주범 | Fabrication method for lightpath modulation device |
US5789168A (en) | 1996-05-01 | 1998-08-04 | Visible Genetics Inc. | Method for amplification and sequencing of nucleic acid polymers |
US5830657A (en) | 1996-05-01 | 1998-11-03 | Visible Genetics Inc. | Method for single-tube sequencing of nucleic acid polymers |
US5795722A (en) | 1997-03-18 | 1998-08-18 | Visible Genetics Inc. | Method and kit for quantitation and nucleic acid sequencing of nucleic acid analytes in a sample |
US20010018514A1 (en) | 1998-07-31 | 2001-08-30 | Mcgall Glenn H. | Nucleic acid labeling compounds |
US6312893B1 (en) | 1996-01-23 | 2001-11-06 | Qiagen Genomics, Inc. | Methods and compositions for determining the sequence of nucleic acid molecules |
TW406461B (en) | 1996-03-01 | 2000-09-21 | Cooper Ind Inc | Enhanced polymer ic weathershed and surge arrester and method of making same |
AU2217597A (en) | 1996-03-18 | 1997-10-22 | Sequenom, Inc. | Dna sequencing by mass spectrometry |
US6361937B1 (en) | 1996-03-19 | 2002-03-26 | Affymetrix, Incorporated | Computer-aided nucleic acid sequencing |
AU2320597A (en) | 1996-03-19 | 1997-10-10 | Molecular Tool, Inc. | Method for determining the nucleotide sequence of a polynucleotide |
SE9601318D0 (en) | 1996-04-04 | 1996-04-04 | Pharmacia Biosensor Ab | Method for nucleic acid analysis |
EP0910664A1 (en) | 1996-04-15 | 1999-04-28 | University Of Southern California | Synthesis of fluorophore-labeled dna |
US5867266A (en) | 1996-04-17 | 1999-02-02 | Cornell Research Foundation, Inc. | Multiple optical channels for chemical analysis |
US6432634B1 (en) | 1996-04-18 | 2002-08-13 | Visible Genetics Inc. | Method, apparatus and kits for sequencing of nucleic acids using multiple dyes |
DK0898596T3 (en) | 1996-04-19 | 2001-12-17 | Amersham Pharm Biotech Uk Ltd | Square type dyes and their use in a sequencing method |
JP3418292B2 (en) | 1996-04-24 | 2003-06-16 | 株式会社日立製作所 | Gene analyzer |
EP0896632B1 (en) | 1996-05-01 | 2001-12-12 | Visible Genetics Inc. | Method for detection and identification of microorganisms |
US5928906A (en) | 1996-05-09 | 1999-07-27 | Sequenom, Inc. | Process for direct sequencing during template amplification |
JP2000512744A (en) | 1996-05-16 | 2000-09-26 | アフィメトリックス,インコーポレイテッド | System and method for detecting label material |
US5846727A (en) | 1996-06-06 | 1998-12-08 | Board Of Supervisors Of Louisiana State University And Agricultural & Mechanical College | Microsystem for rapid DNA sequencing |
JP2000512498A (en) | 1996-06-14 | 2000-09-26 | サルノフ コーポレーション | Polynucleotide sequencing method |
US5771124A (en) | 1996-07-02 | 1998-06-23 | Siliscape | Compact display system with two stage magnification and immersed beam splitter |
WO1998002575A1 (en) | 1996-07-16 | 1998-01-22 | Periannan Senapathy | Method for contiguous genome sequencing |
AU4042597A (en) | 1996-07-19 | 1998-02-10 | Hybridon, Inc. | Method for sequencing nucleic acids using matrix-assisted laser desorption ionization time-of-flight mass spectrometry |
US5922608A (en) | 1996-07-31 | 1999-07-13 | Beckman Instruments, Inc. | Macromolecule sequencing packet and method of use |
US6136212A (en) | 1996-08-12 | 2000-10-24 | The Regents Of The University Of Michigan | Polymer-based micromachining for microfluidic devices |
EP0927268A1 (en) | 1996-08-27 | 1999-07-07 | Visible Genetics Inc. | Apparatus and method for performing sequencing of nucleic acid polymers |
US5832165A (en) | 1996-08-28 | 1998-11-03 | University Of Utah Research Foundation | Composite waveguide for solid phase binding assays |
US6221654B1 (en) | 1996-09-25 | 2001-04-24 | California Institute Of Technology | Method and apparatus for analysis and sorting of polynucleotides based on size |
GB9620209D0 (en) | 1996-09-27 | 1996-11-13 | Cemu Bioteknik Ab | Method of sequencing DNA |
US6020457A (en) | 1996-09-30 | 2000-02-01 | Dendritech Inc. | Disulfide-containing dendritic polymers |
EP0838850A3 (en) | 1996-10-24 | 1999-05-06 | Hamamatsu Photonics K.K. | Method for placing flourescent single molecules on surface of substrate and method for visualizing structural defect of surface of substrate |
US5858671A (en) | 1996-11-01 | 1999-01-12 | The University Of Iowa Research Foundation | Iterative and regenerative DNA sequencing method |
US6258533B1 (en) | 1996-11-01 | 2001-07-10 | The University Of Iowa Research Foundation | Iterative and regenerative DNA sequencing method |
US6002471A (en) | 1996-11-04 | 1999-12-14 | California Institute Of Technology | High resolution scanning raman microscope |
AU746737B2 (en) | 1996-11-06 | 2002-05-02 | Sequenom, Inc. | Compositions and methods for immobilizing nucleic acids to solid supports |
EP1164203B1 (en) | 1996-11-06 | 2007-10-10 | Sequenom, Inc. | DNA Diagnostics based on mass spectrometry |
DE69727489T2 (en) | 1996-11-06 | 2004-11-25 | Sequenom, Inc., San Diego | METHOD OF MASS SPECTROMETRY |
US6133436A (en) | 1996-11-06 | 2000-10-17 | Sequenom, Inc. | Beads bound to a solid support and to nucleic acids |
US6024925A (en) | 1997-01-23 | 2000-02-15 | Sequenom, Inc. | Systems and methods for preparing low volume analyte array elements |
US6140053A (en) | 1996-11-06 | 2000-10-31 | Sequenom, Inc. | DNA sequencing by mass spectrometry via exonuclease degradation |
US6225062B1 (en) | 1996-11-12 | 2001-05-01 | Visible Genetics Inc. | Method and kit for direct isothermal sequencing of nucleic acids |
US5971355A (en) | 1996-11-27 | 1999-10-26 | Xerox Corporation | Microdevice valve structures to fluid control |
US5958703A (en) | 1996-12-03 | 1999-09-28 | Glaxo Group Limited | Use of modified tethers in screening compound libraries |
US6017702A (en) | 1996-12-05 | 2000-01-25 | The Perkin-Elmer Corporation | Chain-termination type nucleic acid sequencing method including 2'-deoxyuridine-5'-triphosphate |
US5876934A (en) | 1996-12-18 | 1999-03-02 | Pharmacia Biotech Inc. | DNA sequencing method |
DE19653439A1 (en) | 1996-12-20 | 1998-07-02 | Svante Dr Paeaebo | Methods for the direct, exponential amplification and sequencing of DNA molecules and their application |
DE19653494A1 (en) | 1996-12-20 | 1998-06-25 | Svante Dr Paeaebo | Process for decoupled, direct, exponential amplification and sequencing of DNA molecules with the addition of a second thermostable DNA polymerase and its application |
US6828094B2 (en) | 1996-12-20 | 2004-12-07 | Roche Diagnostics Gmbh | Method for the uncoupled, direct, exponential amplification and sequencing of DNA molecules with the addition of a second thermostable DNA polymerase and its application |
GB9626815D0 (en) | 1996-12-23 | 1997-02-12 | Cemu Bioteknik Ab | Method of sequencing DNA |
US6046005A (en) | 1997-01-15 | 2000-04-04 | Incyte Pharmaceuticals, Inc. | Nucleic acid sequencing with solid phase capturable terminators comprising a cleavable linking group |
US6309824B1 (en) | 1997-01-16 | 2001-10-30 | Hyseq, Inc. | Methods for analyzing a target nucleic acid using immobilized heterogeneous mixtures of oligonucleotide probes |
US6136543A (en) | 1997-01-31 | 2000-10-24 | Hitachi, Ltd. | Method for determining nucleic acids base sequence and apparatus therefor |
US6403311B1 (en) | 1997-02-12 | 2002-06-11 | Us Genomics | Methods of analyzing polymers using ordered label strategies |
CA2281205A1 (en) | 1997-02-12 | 1998-08-13 | Eugene Y. Chan | Methods and products for analyzing polymers |
US6117973A (en) | 1997-02-24 | 2000-09-12 | Georgia Tech Research Corp. | PNA monomers with electron donor or acceptor |
US5837860A (en) | 1997-03-05 | 1998-11-17 | Molecular Tool, Inc. | Covalent attachment of nucleic acid molecules onto solid-phases via disulfide bonds |
US6117634A (en) | 1997-03-05 | 2000-09-12 | The Reagents Of The University Of Michigan | Nucleic acid sequencing and mapping |
WO1998040223A1 (en) | 1997-03-11 | 1998-09-17 | Polaroid Corporation | Substrate with non-visible indicium |
WO1998040520A1 (en) | 1997-03-14 | 1998-09-17 | Hybridon, Inc. | Method for sequencing of modified nucleic acids using electrospray ionization-fourier transform mass spectrometry |
US7144699B2 (en) | 1997-03-20 | 2006-12-05 | Affymetrix, Inc. | Iterative resequencing |
ATE269908T1 (en) | 1997-04-01 | 2004-07-15 | Manteia S A | METHOD FOR SEQUENCING NUCLEIC ACIDS |
US6391622B1 (en) | 1997-04-04 | 2002-05-21 | Caliper Technologies Corp. | Closed-loop biochemical analyzers |
EP0972082A4 (en) | 1997-04-04 | 2007-04-25 | Caliper Life Sciences Inc | Closed-loop biochemical analyzers |
US6235471B1 (en) | 1997-04-04 | 2001-05-22 | Caliper Technologies Corp. | Closed-loop biochemical analyzers |
EP0985142A4 (en) | 1997-05-23 | 2006-09-13 | Lynx Therapeutics Inc | System and apparaus for sequential processing of analytes |
US6969488B2 (en) | 1998-05-22 | 2005-11-29 | Solexa, Inc. | System and apparatus for sequential processing of analytes |
US5945284A (en) | 1997-05-27 | 1999-08-31 | The Perkin-Elmer Corporation | Length determination of nucleic acid repeat sequences by discontinuous primer extension |
SE9702008D0 (en) | 1997-05-28 | 1997-05-28 | Pharmacia Biotech Ab | A method and a system for nucleic acid seouence analysis |
US5919626A (en) | 1997-06-06 | 1999-07-06 | Orchid Bio Computer, Inc. | Attachment of unmodified nucleic acids to silanized solid phase surfaces |
AU8224998A (en) | 1997-07-04 | 1999-01-25 | Nycomed Amersham Plc | Peroxidase-catalysed fluorescence |
WO1999005221A1 (en) | 1997-07-28 | 1999-02-04 | Nycomed Amersham Plc | Cyanine dyes |
PT1017848E (en) | 1997-07-28 | 2003-02-28 | Medical Biosystems Ltd | ANALYSIS OF NUCLEIC ACID SEQUENCES |
US6245506B1 (en) | 1997-07-30 | 2001-06-12 | Bbi Bioseq, Inc. | Integrated sequencing device |
GB9716231D0 (en) | 1997-07-31 | 1997-10-08 | Amersham Int Ltd | Base analogues |
US5882904A (en) | 1997-08-04 | 1999-03-16 | Amersham Pharmacia Biotech Inc. | Thermococcus barossii DNA polymerase mutants |
BR9811158A (en) | 1997-08-12 | 2000-07-25 | Eastman Chem Co | Alkyd modified by acrylic, water-based latex, preparation process, and coating composition |
US5994085A (en) | 1997-08-26 | 1999-11-30 | Cantor; Thomas L. | Methods and devices for detecting non-complexed prostate specific antigen |
US6087099A (en) | 1997-09-08 | 2000-07-11 | Myriad Genetics, Inc. | Method for sequencing both strands of a double stranded DNA in a single sequencing reaction |
US6346379B1 (en) | 1997-09-11 | 2002-02-12 | F. Hoffman-La Roche Ag | Thermostable DNA polymerases incorporating nucleoside triphosphates labeled with fluorescein family dyes |
WO1999013110A1 (en) | 1997-09-11 | 1999-03-18 | Seq, Ltd. | Method to make fluorescent nucleotide photoproducts for dna sequencing and analysis |
AU9476298A (en) | 1997-09-11 | 1999-03-29 | Seq, Ltd. | Method for dna sequencing analysis |
TW352471B (en) | 1997-09-20 | 1999-02-11 | United Microelectronics Corp | Method for preventing B-P-Si glass from subsiding |
US5836750A (en) | 1997-10-09 | 1998-11-17 | Honeywell Inc. | Electrostatically actuated mesopump having a plurality of elementary cells |
US6485944B1 (en) | 1997-10-10 | 2002-11-26 | President And Fellows Of Harvard College | Replica amplification of nucleic acid arrays |
US6511803B1 (en) | 1997-10-10 | 2003-01-28 | President And Fellows Of Harvard College | Replica amplification of nucleic acid arrays |
US5958694A (en) | 1997-10-16 | 1999-09-28 | Caliper Technologies Corp. | Apparatus and methods for sequencing nucleic acids in microfluidic systems |
US6049380A (en) | 1997-11-12 | 2000-04-11 | Regents Of The University Of California | Single molecule identification using selected fluorescence characteristics |
US6322968B1 (en) | 1997-11-21 | 2001-11-27 | Orchid Biosciences, Inc. | De novo or “universal” sequencing array |
US6268131B1 (en) | 1997-12-15 | 2001-07-31 | Sequenom, Inc. | Mass spectrometric methods for sequencing nucleic acids |
US6407858B1 (en) | 1998-05-14 | 2002-06-18 | Genetic Microsystems, Inc | Focusing of microscopes and reading of microarrays |
US6262838B1 (en) | 1998-03-20 | 2001-07-17 | Genetic Microsystems Inc | Focusing in microscope systems |
US6269846B1 (en) | 1998-01-13 | 2001-08-07 | Genetic Microsystems, Inc. | Depositing fluid specimens on substrates, resulting ordered arrays, techniques for deposition of arrays |
JP4317953B2 (en) | 1998-01-22 | 2009-08-19 | 独立行政法人理化学研究所 | DNA sequence determination method |
WO1999037810A1 (en) | 1998-01-23 | 1999-07-29 | Amersham Pharmacia Biotech, Inc. | A method, reagent solution and kits for dna sequencing |
US6280954B1 (en) | 1998-02-02 | 2001-08-28 | Amersham Pharmacia Biotech Ab | Arrayed primer extension technique for nucleic acid analysis |
JP4306960B2 (en) | 1998-02-04 | 2009-08-05 | ジーイー・ヘルスケア・バイオサイエンス・コーポレイション | Dideoxy dye terminator |
US6150147A (en) | 1998-02-06 | 2000-11-21 | Affymetrix, Inc. | Biological array fabrication methods with reduction of static charge |
IL123256A0 (en) | 1998-02-10 | 1998-09-24 | Yeda Res & Dev | Methods for dna amplification and sequencing |
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 |
CA2324248A1 (en) | 1998-03-18 | 1999-09-23 | Amersham Pharmacia Biotech Inc. | Thermostable dna polymerase from thermoanaerobacter thermohydrosulfuricus |
GB9805918D0 (en) | 1998-03-19 | 1998-05-13 | Nycomed Amersham Plc | Sequencing by hybridisation |
US6185030B1 (en) | 1998-03-20 | 2001-02-06 | James W. Overbeck | Wide field of view and high speed scanning microscopy |
US6719868B1 (en) | 1998-03-23 | 2004-04-13 | President And Fellows Of Harvard College | Methods for fabricating microfluidic structures |
AU3199699A (en) | 1998-03-23 | 1999-10-18 | Invitrogen Corporation | Modified nucleotides and methods useful for nucleic acid sequencing |
DE29806082U1 (en) | 1998-04-02 | 1998-06-18 | Ideal Electronics Inc | Cooling device for a central processing unit |
CA2328881A1 (en) | 1998-04-16 | 1999-10-21 | Northeastern University | Expert system for analysis of dna sequencing electropherograms |
US5945325A (en) | 1998-04-20 | 1999-08-31 | California Institute Of Technology | Thermally stable para-nitrobenzyl esterases |
EP1082458A1 (en) | 1998-05-01 | 2001-03-14 | Arizona Board Of Regents | Method of determining the nucleotide sequence of oligonucleotides and dna molecules |
US6780591B2 (en) | 1998-05-01 | 2004-08-24 | Arizona Board Of Regents | Method of determining the nucleotide sequence of oligonucleotides and DNA molecules |
US6130046A (en) | 1998-05-04 | 2000-10-10 | Affymetrix, Inc. | Techniques for synthesis integrity evaluation utilizing cycle fidelity probes |
US6261848B1 (en) | 1998-05-08 | 2001-07-17 | The Johns Hopkins University | Miniature immuno-optical rapid analyte sensor platform |
ATE530891T1 (en) | 1998-05-22 | 2011-11-15 | California Inst Of Techn | MINIATURIZED CELL SORTER |
WO1999065076A1 (en) | 1998-06-05 | 1999-12-16 | Hitachi, Ltd. | Semiconductor device and method for manufacturing the same |
JPH11352409A (en) | 1998-06-05 | 1999-12-24 | Olympus Optical Co Ltd | Fluorescence detector |
WO1999064840A1 (en) | 1998-06-09 | 1999-12-16 | Caliper Technologies Corp. | Fluorescent polarization detection in microfluidic systems |
US6287821B1 (en) | 1998-06-11 | 2001-09-11 | Orchid Biosciences, Inc. | Nucleotide analogues with 3'-pro-fluorescent fluorophores in nucleic acid sequence analysis |
US6872521B1 (en) | 1998-06-16 | 2005-03-29 | Beckman Coulter, Inc. | Polymerase signaling assay |
EP1086209B1 (en) | 1998-06-17 | 2007-08-22 | Amersham Biosciences Corp. | Fy7 polymerase |
GB9813216D0 (en) | 1998-06-18 | 1998-08-19 | Pyrosequencing Ab | Reaction monitoring systems |
US6404907B1 (en) | 1998-06-26 | 2002-06-11 | Visible Genetics Inc. | Method for sequencing nucleic acids with reduced errors |
US6235473B1 (en) | 1998-07-02 | 2001-05-22 | Orchid Biosciences, Inc. | Gene pen devices for array printing |
US7399844B2 (en) | 1998-07-09 | 2008-07-15 | Agilent Technologies, Inc. | Method and reagents for analyzing the nucleotide sequence of nucleic acids |
WO2000006770A1 (en) | 1998-07-30 | 2000-02-10 | Solexa Ltd. | Arrayed biomolecules and their use in sequencing |
US20030022207A1 (en) * | 1998-10-16 | 2003-01-30 | Solexa, Ltd. | Arrayed polynucleotides and their use in genome analysis |
US20040106110A1 (en) | 1998-07-30 | 2004-06-03 | Solexa, Ltd. | Preparation of polynucleotide arrays |
US6787308B2 (en) | 1998-07-30 | 2004-09-07 | Solexa Ltd. | Arrayed biomolecules and their use in sequencing |
GB0002310D0 (en) | 2000-02-01 | 2000-03-22 | Solexa Ltd | Polynucleotide sequencing |
US6447724B1 (en) | 1998-08-11 | 2002-09-10 | Caliper Technologies Corp. | DNA sequencing using multiple fluorescent labels being distinguishable by their decay times |
WO2000009753A1 (en) | 1998-08-11 | 2000-02-24 | Caliper Technologies Corp. | Methods and systems for sequencing dna by distinguishing the decay times of fluorescent probes |
US6716394B2 (en) | 1998-08-11 | 2004-04-06 | Caliper Technologies Corp. | DNA sequencing using multiple fluorescent labels being distinguishable by their decay times |
US6210896B1 (en) | 1998-08-13 | 2001-04-03 | Us Genomics | Molecular motors |
US6263286B1 (en) | 1998-08-13 | 2001-07-17 | U.S. Genomics, Inc. | Methods of analyzing polymers using a spatial network of fluorophores and fluorescence resonance energy transfer |
US6245507B1 (en) | 1998-08-18 | 2001-06-12 | Orchid Biosciences, Inc. | In-line complete hyperspectral fluorescent imaging of nucleic acid molecules |
US6306643B1 (en) | 1998-08-24 | 2001-10-23 | Affymetrix, Inc. | Methods of using an array of pooled probes in genetic analysis |
AU9505798A (en) | 1998-09-24 | 2000-04-10 | Biotraces, Inc. | Sequencing duplex dna by mass spectroscopy |
AU6504099A (en) | 1998-09-29 | 2000-04-17 | Diversa Corporation | Nucleic acids and proteins from Cenarchaeum symbiosum |
DE19844931C1 (en) * | 1998-09-30 | 2000-06-15 | Stefan Seeger | Procedures for DNA or RNA sequencing |
US6458945B1 (en) | 1998-10-01 | 2002-10-01 | Variagenics, Inc. | Method for analyzing polynucleotides |
US6566059B1 (en) | 1998-10-01 | 2003-05-20 | Variagenics, Inc. | Method for analyzing polynucleotides |
US6221592B1 (en) | 1998-10-20 | 2001-04-24 | Wisconsin Alumi Research Foundation | Computer-based methods and systems for sequencing of individual nucleic acid molecules |
US6607888B2 (en) | 1998-10-20 | 2003-08-19 | Wisconsin Alumni Research Foundation | Method for analyzing nucleic acid reactions |
DE19849348A1 (en) | 1998-10-26 | 2000-04-27 | Univ Ludwigs Albert | Identification and/or sequencing of an unknown DNA or RNA sequence adjacent to a known DNA or RNA region comprises linker-mediated PCR following amplification by linear PCR |
US6545264B1 (en) | 1998-10-30 | 2003-04-08 | Affymetrix, Inc. | Systems and methods for high performance scanning |
RU2143343C1 (en) | 1998-11-03 | 1999-12-27 | Самсунг Электроникс Ко., Лтд. | Microinjector and microinjector manufacture method |
US6309701B1 (en) | 1998-11-10 | 2001-10-30 | Bio-Pixels Ltd. | Fluorescent nanocrystal-labeled microspheres for fluorescence analyses |
US6387982B1 (en) | 1998-11-23 | 2002-05-14 | Dentsply Detrey G.M.B.H. | Self etching adhesive primer composition and polymerizable surfactants |
US6245518B1 (en) | 1998-12-11 | 2001-06-12 | Hyseq, Inc. | Polynucleotide arrays and methods of making and using the same |
DE69930310T3 (en) | 1998-12-14 | 2009-12-17 | Pacific Biosciences of California, Inc. (n. d. Ges. d. Staates Delaware), Menlo Park | KIT AND METHOD FOR THE NUCLEIC ACID SEQUENCING OF INDIVIDUAL MOLECULES BY POLYMERASE SYNTHESIS |
EP1141402A4 (en) | 1998-12-18 | 2004-10-06 | Univ California | Method for the detection of specific nucleic acid sequences by polymerase nucleotide incorporation |
US6340750B1 (en) | 1998-12-18 | 2002-01-22 | The Texas A&M University System | Through bond energy transfer in fluorescent dyes for labelling biological molecules |
GB9828785D0 (en) | 1998-12-30 | 1999-02-17 | Amersham Pharm Biotech Ab | Sequencing systems |
DE60042775D1 (en) | 1999-01-06 | 2009-10-01 | Callida Genomics Inc | IMPROVED SEQUENCING BY HYBRIDIZATION THROUGH THE USE OF PROBABLE MIXTURES |
US6361671B1 (en) | 1999-01-11 | 2002-03-26 | The Regents Of The University Of California | Microfabricated capillary electrophoresis chip and method for simultaneously detecting multiple redox labels |
WO2000042223A1 (en) | 1999-01-15 | 2000-07-20 | Myriad Genetics, Inc. | Method for controlling the distribution of dna sequencing termination products |
GB9901475D0 (en) | 1999-01-22 | 1999-03-17 | Pyrosequencing Ab | A method of DNA sequencing |
US6270644B1 (en) | 1999-01-27 | 2001-08-07 | Affymetrix, Inc. | Capillary array electrophoresis scanner |
EP1163052B1 (en) | 1999-02-23 | 2010-06-02 | Caliper Life Sciences, Inc. | Manipulation of microparticles in microfluidic systems |
AU3508600A (en) | 1999-02-26 | 2000-09-14 | Orchid Biosciences, Inc. | Microstructures for use in biological assays and reactions |
US6558945B1 (en) | 1999-03-08 | 2003-05-06 | Aclara Biosciences, Inc. | Method and device for rapid color detection |
EP1159453B1 (en) | 1999-03-10 | 2008-05-28 | ASM Scientific, Inc. | A method for direct nucleic acid sequencing |
EP1192274A2 (en) | 1999-03-25 | 2002-04-03 | Hyseq, Inc. | Solution-based methods and materials for sequence analysis by hybridization |
EP1165839A2 (en) | 1999-03-26 | 2002-01-02 | Whitehead Institute For Biomedical Research | Universal arrays |
US6403317B1 (en) | 1999-03-26 | 2002-06-11 | Affymetrix, Inc. | Electronic detection of hybridization on nucleic acid arrays |
AU3567900A (en) | 1999-03-30 | 2000-10-16 | Solexa Ltd. | Polynucleotide sequencing |
US6261775B1 (en) | 1999-04-09 | 2001-07-17 | The Regents Of The University Of California | Detection of chromosome copy number changes to distinguish melanocytic nevi from malignant melanoma |
US6573047B1 (en) | 1999-04-13 | 2003-06-03 | Dna Sciences, Inc. | Detection of nucleotide sequence variation through fluorescence resonance energy transfer label generation |
US6368562B1 (en) | 1999-04-16 | 2002-04-09 | Orchid Biosciences, Inc. | Liquid transportation system for microfluidic device |
US20030108867A1 (en) | 1999-04-20 | 2003-06-12 | Chee Mark S | Nucleic acid sequencing using microsphere arrays |
US6521428B1 (en) | 1999-04-21 | 2003-02-18 | Genome Technologies, Llc | Shot-gun sequencing and amplification without cloning |
US20020142329A1 (en) | 1999-04-30 | 2002-10-03 | Aclara Biosciences, Inc. | Compositions and methods employing cleavable electrophoretic tag reagents |
US6673550B2 (en) | 1999-04-30 | 2004-01-06 | Aclara Biosciences, Inc. | Electrophoretic tag reagents comprising fluorescent compounds |
US6395559B1 (en) | 1999-05-04 | 2002-05-28 | Orchid Biosciences, Inc. | Multiple fluid sample processor with single well addressability |
US7655443B1 (en) | 1999-05-07 | 2010-02-02 | Siemens Healthcare Diagnostics, Inc. | Nucleic acid sequencing with simultaneous quantitation |
US6423273B1 (en) | 1999-05-19 | 2002-07-23 | Orchid Biosciences, Inc. | Method of forming seals for a microfluidic device |
US7056661B2 (en) | 1999-05-19 | 2006-06-06 | Cornell Research Foundation, Inc. | Method for sequencing nucleic acid molecules |
US6472141B2 (en) | 1999-05-21 | 2002-10-29 | Caliper Technologies Corp. | Kinase assays using polycations |
US6515120B1 (en) | 1999-05-25 | 2003-02-04 | Praelux Incorporated | Method for sequencing and characterizing polymeric biomolecules using aptamers and a method for producing aptamers |
US6485690B1 (en) | 1999-05-27 | 2002-11-26 | Orchid Biosciences, Inc. | Multiple fluid sample processor and system |
US6225109B1 (en) | 1999-05-27 | 2001-05-01 | Orchid Biosciences, Inc. | Genetic analysis device |
US6309886B1 (en) | 1999-06-04 | 2001-10-30 | The Regents Of The University Of California | High throughput analysis of samples in flowing liquid |
WO2000079007A1 (en) | 1999-06-19 | 2000-12-28 | Hyseq Inc. | Improved methods of sequence assembly in sequencing by hybridization |
CA2721172C (en) | 1999-06-28 | 2012-04-10 | California Institute Of Technology | Microfabricated elastomeric valve and pump systems |
US6444106B1 (en) | 1999-07-09 | 2002-09-03 | Orchid Biosciences, Inc. | Method of moving fluid in a microfluidic device |
US6444173B1 (en) | 1999-07-09 | 2002-09-03 | Orchid Biosciences, Inc. | Method of moving and detecting fluid in a microfluidic device |
US6448090B1 (en) | 1999-07-09 | 2002-09-10 | Orchid Biosciences, Inc. | Fluid delivery system for a microfluidic device using alternating pressure waveforms |
US6268219B1 (en) | 1999-07-09 | 2001-07-31 | Orchid Biosciences, Inc. | Method and apparatus for distributing fluid in a microfluidic device |
US6395232B1 (en) | 1999-07-09 | 2002-05-28 | Orchid Biosciences, Inc. | Fluid delivery system for a microfluidic device using a pressure pulse |
WO2001004357A2 (en) | 1999-07-13 | 2001-01-18 | Whitehead Institute For Biomedical Research | Generic sbe-fret protocol |
US20020045182A1 (en) | 1999-07-16 | 2002-04-18 | Lynx Therapeutics, Inc. | Multiplexed differential displacement for nucleic acid determinations |
US6927065B2 (en) | 1999-08-13 | 2005-08-09 | U.S. Genomics, Inc. | Methods and apparatus for characterization of single polymers |
AU7086800A (en) | 1999-08-30 | 2001-03-26 | Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services, The | High speed parallel molecular nucleic acid sequencing |
US6528258B1 (en) | 1999-09-03 | 2003-03-04 | Lifebeam Technologies, Inc. | Nucleic acid sequencing using an optically labeled pore |
US7244559B2 (en) | 1999-09-16 | 2007-07-17 | 454 Life Sciences Corporation | Method of sequencing a nucleic acid |
US6274320B1 (en) | 1999-09-16 | 2001-08-14 | Curagen Corporation | Method of sequencing a nucleic acid |
US6107061A (en) | 1999-09-18 | 2000-08-22 | The Perkin-Elmer Corporation | Modified primer extension reactions for polynucleotide sequence detection |
WO2001023610A2 (en) | 1999-09-29 | 2001-04-05 | Solexa Ltd. | Polynucleotide sequencing |
GB9923324D0 (en) | 1999-10-01 | 1999-12-08 | Pyrosequencing Ab | Separation apparatus and method |
US6309836B1 (en) | 1999-10-05 | 2001-10-30 | Marek Kwiatkowski | Compounds for protecting hydroxyls and methods for their use |
US6908736B1 (en) | 1999-10-06 | 2005-06-21 | Medical Biosystems, Ltd. | DNA sequencing method |
GB9923644D0 (en) | 1999-10-06 | 1999-12-08 | Medical Biosystems Ltd | DNA sequencing |
US6077674A (en) | 1999-10-27 | 2000-06-20 | Agilent Technologies Inc. | Method of producing oligonucleotide arrays with features of high purity |
EP1096024A1 (en) | 1999-10-28 | 2001-05-02 | Remacle, José | Method and kit for the screening and/or the quantification of multiple homologous nucleic acid sequences on arrays |
WO2001032930A1 (en) | 1999-11-04 | 2001-05-10 | California Institute Of Technology | Methods and apparatuses for analyzing polynucleotide sequences |
WO2001038546A1 (en) | 1999-11-23 | 2001-05-31 | Amersham Biosciences Corp | Improving dideoxynucleotide-triphosphate utilization by the hyper-thermophilic dna polymerase from the archaeon pyrococcus furiosus |
CN1336960A (en) | 1999-11-26 | 2002-02-20 | 株式会社百尼尔 | DNA sequencing method which employs various DNA polymerases and kit used for the same |
US6383749B2 (en) * | 1999-12-02 | 2002-05-07 | Clontech Laboratories, Inc. | Methods of labeling nucleic acids for use in array based hybridization assays |
US6384487B1 (en) | 1999-12-06 | 2002-05-07 | Micron Technology, Inc. | Bow resistant plastic semiconductor package and method of fabrication |
GB9929381D0 (en) | 1999-12-10 | 2000-02-09 | Pyrosequencing Ab | A method of assessing the amount of nucleic acid in a sample |
WO2001048184A2 (en) | 1999-12-23 | 2001-07-05 | Axaron Bioscience Ag | Method for carrying out the parallel sequencing of a nucleic acid mixture on a surface |
GB0002389D0 (en) | 2000-02-02 | 2000-03-22 | Solexa Ltd | Molecular arrays |
EP1198596A1 (en) | 2000-02-15 | 2002-04-24 | Lynx Therapeutics, Inc. | Data analysis and display system for ligation-based dna sequencing |
US6632645B1 (en) | 2000-03-02 | 2003-10-14 | Promega Corporation | Thermophilic DNA polymerases from Thermoactinomyces vulgaris |
JP3442338B2 (en) | 2000-03-17 | 2003-09-02 | 株式会社日立製作所 | DNA analyzer, DNA base sequencer, DNA base sequence determination method, and reaction module |
WO2001075154A2 (en) | 2000-04-03 | 2001-10-11 | Axaron Bioscience Ag | Novel method for the parallel sequencing of a nucleic acid mixture on a surface |
US6436641B1 (en) | 2000-04-17 | 2002-08-20 | Visible Genetics Inc. | Method and apparatus for DNA sequencing |
AU2001270504A1 (en) | 2000-05-04 | 2001-11-12 | Syngenta Participations Ag | Novel assay for nucleic acid analysis |
US6342326B1 (en) | 2000-05-10 | 2002-01-29 | Beckman Coulter, Inc. | Synthesis and use of acyl fluorides of cyanine dyes |
AU2001262284A1 (en) | 2000-05-12 | 2001-11-20 | Gnothis Holding Sa | Method for detecting polynucleotides using fluorescence correlation spectroscopy |
GB0013276D0 (en) | 2000-06-01 | 2000-07-26 | Amersham Pharm Biotech Uk Ltd | Nucleotide analogues |
US20020168678A1 (en) | 2000-06-07 | 2002-11-14 | Li-Cor, Inc. | Flowcell system for nucleic acid sequencing |
US6869764B2 (en) | 2000-06-07 | 2005-03-22 | L--Cor, Inc. | Nucleic acid sequencing using charge-switch nucleotides |
ATE423221T1 (en) | 2000-06-13 | 2009-03-15 | Univ Boston | USE OF MASS-MATCHED NUCLEOTIDES IN THE ANALYSIS OF OLIGONUCLEOTIDE MIXTURES AND IN HIGH-MULTIPLEX NUCLEIC ACID SEQUENCING |
US6829753B2 (en) | 2000-06-27 | 2004-12-07 | Fluidigm Corporation | Microfluidic design automation method and system |
DE10031842A1 (en) | 2000-06-30 | 2002-01-31 | Gnothis Holding Sa Ecublens | Multiplex sequencing method |
GB0016258D0 (en) | 2000-07-03 | 2000-08-23 | Amersham Pharm Biotech Uk Ltd | Base analogues |
GB0016473D0 (en) | 2000-07-05 | 2000-08-23 | Amersham Pharm Biotech Uk Ltd | Sequencing method |
GB0016472D0 (en) | 2000-07-05 | 2000-08-23 | Amersham Pharm Biotech Uk Ltd | Sequencing method and apparatus |
CN101525660A (en) * | 2000-07-07 | 2009-09-09 | 维西根生物技术公司 | An instant sequencing methodology |
US20030017461A1 (en) | 2000-07-11 | 2003-01-23 | Aclara Biosciences, Inc. | Tag cleavage for detection of nucleic acids |
US6397150B1 (en) | 2000-07-27 | 2002-05-28 | Visible Genetics Inc. | Method and apparatus for sequencing of DNA using an internal calibrant |
JP2004512533A (en) | 2000-08-22 | 2004-04-22 | アフィメトリックス インコーポレイテッド | Systems, methods and computer software products for controlling biological microarray scanners |
GB0021977D0 (en) | 2000-09-07 | 2000-10-25 | Pyrosequencing Ab | Method of sequencing DNA |
GB0022069D0 (en) | 2000-09-08 | 2000-10-25 | Pyrosequencing Ab | Method |
US6627748B1 (en) | 2000-09-11 | 2003-09-30 | The Trustees Of Columbia University In The City Of New York | Combinatorial fluorescence energy transfer tags and their applications for multiplex genetic analyses |
JP3638516B2 (en) | 2000-09-28 | 2005-04-13 | 株式会社日立製作所 | Nucleic acid detection method and nucleic acid detection kit |
US6835537B1 (en) | 2000-09-29 | 2004-12-28 | Myriad Genetics, Inc. | Method for equalizing band intensities on sequencing gels |
US7258774B2 (en) | 2000-10-03 | 2007-08-21 | California Institute Of Technology | Microfluidic devices and methods of use |
AU2001296645A1 (en) | 2000-10-06 | 2002-04-15 | The Trustees Of Columbia University In The City Of New York | Massive parallel method for decoding dna and rna |
WO2002029113A2 (en) * | 2000-10-06 | 2002-04-11 | Novozymes Biotech, Inc. | Methods for monitoring multiple gene expression |
AU1189702A (en) | 2000-10-13 | 2002-04-22 | Fluidigm Corp | Microfluidic device based sample injection system for analytical devices |
US6448407B1 (en) | 2000-11-01 | 2002-09-10 | Pe Corporation (Ny) | Atropisomers of asymmetric xanthene fluorescent dyes and methods of DNA sequencing and fragment analysis |
US20020115092A1 (en) | 2000-11-08 | 2002-08-22 | The Scripps Research Institute | Energy transfer labels with mechanically linked fluorophores |
US6770472B2 (en) | 2000-11-17 | 2004-08-03 | The Board Of Trustees Of The Leland Stanford Junior University | Direct DNA sequencing with a transcription protein and a nanometer scale electrometer |
AU2002217784A1 (en) | 2000-11-28 | 2002-06-11 | Promega Corporation | Purification of dna sequencing reactions using silica magnetic particles |
WO2002055997A2 (en) | 2001-01-12 | 2002-07-18 | Karolinska Innovations Ab | Substrate for fluorescence analysis |
US20020197618A1 (en) | 2001-01-20 | 2002-12-26 | Sampson Jeffrey R. | Synthesis and amplification of unstructured nucleic acids for rapid sequencing |
US20020102595A1 (en) | 2001-01-29 | 2002-08-01 | Davis Lloyd Mervyn | Methods for detection of incorporation of a nucleotide onto a nucleic acid primer |
AU2002231934A1 (en) | 2001-01-30 | 2002-08-12 | Solexa Ltd. | Arrayed polynucleotides and their use in genome analysis |
US7026163B1 (en) | 2001-02-23 | 2006-04-11 | Mayo Foundation For Medical Education And Research | Sulfotransferase sequence variants |
CA2440615A1 (en) | 2001-03-12 | 2002-09-19 | Affymetrix, Inc. | Nucleic acid labeling compounds |
US20040038206A1 (en) * | 2001-03-14 | 2004-02-26 | Jia Zhang | Method for high throughput assay of genetic analysis |
DE10115309A1 (en) | 2001-03-28 | 2002-10-02 | Gnothis Holding Sa Ecublens | Microscope arrangement for fluorescence spectroscopy, in particular fluorescence correlation spectroscopy |
US20030027140A1 (en) | 2001-03-30 | 2003-02-06 | Jingyue Ju | High-fidelity DNA sequencing using solid phase capturable dideoxynucleotides and mass spectrometry |
DE10120798B4 (en) | 2001-04-27 | 2005-12-29 | Genovoxx Gmbh | Method for determining gene expression |
DE10120797B4 (en) | 2001-04-27 | 2005-12-22 | Genovoxx Gmbh | Method for analyzing nucleic acid chains |
CN1384203A (en) | 2001-04-30 | 2002-12-11 | 香港科技创业股份有限公司 | Low temperature circulating DNA extending reaction method with high extension specificity |
US20020172948A1 (en) | 2001-05-04 | 2002-11-21 | Perlin Mark W. | Method and system for nucleic acid sequencing |
US20030211486A1 (en) | 2001-05-25 | 2003-11-13 | Frudakis Tony N. | Compositions and methods for detecting polymorphisms associated with pigmentation |
US20050260570A1 (en) | 2001-05-29 | 2005-11-24 | Mao Jen-I | Sequencing by proxy |
US7118907B2 (en) | 2001-06-06 | 2006-10-10 | Li-Cor, Inc. | Single molecule detection systems and methods |
US6689478B2 (en) * | 2001-06-21 | 2004-02-10 | Corning Incorporated | Polyanion/polycation multilayer film for DNA immobilization |
US6613523B2 (en) | 2001-06-29 | 2003-09-02 | Agilent Technologies, Inc. | Method of DNA sequencing using cleavable tags |
GB0119719D0 (en) | 2001-08-13 | 2001-10-03 | Solexa Ltd | DNA sequence analysis |
US7172865B2 (en) * | 2001-08-13 | 2007-02-06 | Saint Louis University | Rapid and sensitive assay for the detection and quantification of coregulators of nucleic acid binding factors |
US6995841B2 (en) | 2001-08-28 | 2006-02-07 | Rice University | Pulsed-multiline excitation for color-blind fluorescence detection |
WO2003020968A2 (en) * | 2001-08-29 | 2003-03-13 | Genovoxx Gmbh | Method for analyzing nucleic acid sequences and gene expression |
US7223541B2 (en) | 2001-08-29 | 2007-05-29 | Ge Healthcare Bio-Sciences Corp. | Terminal-phosphate-labeled nucleotides and methods of use |
US20030104386A1 (en) | 2001-08-31 | 2003-06-05 | The Regents Of The University Of California | Methods for the specific detection of redox-active tags and the use thereof for capillary gel electrophoresis and DNA sequencing |
DE60223276T2 (en) * | 2001-08-31 | 2008-08-14 | Datascope Investment Corp. | Method for blocking nonspecific hybridizations of nucleic acid sequences |
US6852492B2 (en) | 2001-09-24 | 2005-02-08 | Intel Corporation | Nucleic acid sequencing by raman monitoring of uptake of precursors during molecular replication |
US6975943B2 (en) | 2001-09-24 | 2005-12-13 | Seqwright, Inc. | Clone-array pooled shotgun strategy for nucleic acid sequencing |
US6982165B2 (en) | 2001-09-24 | 2006-01-03 | Intel Corporation | Nucleic acid sequencing by raman monitoring of molecular deconstruction |
DE10246005A1 (en) | 2001-10-04 | 2003-04-30 | Genovoxx Gmbh | Automated nucleic acid sequencer, useful e.g. for analyzing gene expression, based on parallel incorporation of fluorescently labeled terminating nucleotides |
US7406385B2 (en) * | 2001-10-25 | 2008-07-29 | Applera Corporation | System and method for consensus-calling with per-base quality values for sample assemblies |
US20040054162A1 (en) | 2001-10-30 | 2004-03-18 | Hanna Michelle M. | Molecular detection systems utilizing reiterative oligonucleotide synthesis |
WO2003044678A1 (en) | 2001-11-22 | 2003-05-30 | Hitachi, Ltd. | Data processing system using base sequence-related data |
JP4163622B2 (en) | 2001-11-22 | 2008-10-08 | 株式会社日立製作所 | Information processing method, information processing apparatus, information processing program, and recording medium on which information processing program is recorded |
GB0128526D0 (en) | 2001-11-29 | 2002-01-23 | Amersham Pharm Biotech Uk Ltd | Nucleotide analogues |
US7057026B2 (en) | 2001-12-04 | 2006-06-06 | Solexa Limited | Labelled nucleotides |
GB0200938D0 (en) | 2002-01-16 | 2002-03-06 | Solexa Ltd | Prism design for scanning applications |
US7211382B2 (en) | 2002-04-09 | 2007-05-01 | Orchid Cellmark Inc. | Primer extension using modified nucleotides |
EP1497304B1 (en) * | 2002-04-12 | 2014-06-25 | Catalyst Assets LLC | Dual-labeled nucleotides |
US7108976B2 (en) | 2002-06-17 | 2006-09-19 | Affymetrix, Inc. | Complexity management of genomic DNA by locus specific amplification |
DE10256898B4 (en) | 2002-11-29 | 2006-01-12 | Senslab-Gesellschaft Zur Entwicklung Und Herstellung Bioelektrochemischer Sensoren Mbh | Electrochemical detection of nucleic acids |
US6855503B2 (en) * | 2002-12-20 | 2005-02-15 | Amersham Biosciences Corp | Heterocyclic FRETdye cassettes for labeling biological molecules and their use in DNA sequencing |
US8676383B2 (en) | 2002-12-23 | 2014-03-18 | Applied Biosystems, Llc | Device for carrying out chemical or biological reactions |
US6977153B2 (en) | 2002-12-31 | 2005-12-20 | Qiagen Gmbh | Rolling circle amplification of RNA |
WO2004074503A2 (en) | 2003-02-21 | 2004-09-02 | Hoser Mark J | Nucleic acid sequencing methods, kits and reagents |
GB0400584D0 (en) * | 2004-01-12 | 2004-02-11 | Solexa Ltd | Nucleic acid chacterisation |
EP2248911A1 (en) | 2004-02-19 | 2010-11-10 | Helicos Biosciences Corporation | Methods and kits for analyzing polynucleotide sequences |
US20100216153A1 (en) | 2004-02-27 | 2010-08-26 | Helicos Biosciences Corporation | Methods for detecting fetal nucleic acids and diagnosing fetal abnormalities |
US20050239085A1 (en) | 2004-04-23 | 2005-10-27 | Buzby Philip R | Methods for nucleic acid sequence determination |
US7492462B2 (en) * | 2006-01-17 | 2009-02-17 | Honeywell International, Inc. | Optochemical sensor |
US7282337B1 (en) | 2006-04-14 | 2007-10-16 | Helicos Biosciences Corporation | Methods for increasing accuracy of nucleic acid sequencing |
-
2004
- 2004-05-24 US US10/852,482 patent/US7169560B2/en not_active Expired - Fee Related
- 2004-11-12 WO PCT/US2004/037613 patent/WO2005047523A2/en active Application Filing
- 2004-11-12 EP EP04810723A patent/EP1692312A4/en not_active Ceased
- 2004-11-12 CA CA002545619A patent/CA2545619A1/en not_active Abandoned
-
2006
- 2006-10-26 US US11/588,108 patent/US7491498B2/en not_active Expired - Fee Related
-
2009
- 2009-02-13 US US12/371,310 patent/US7897345B2/en not_active Expired - Fee Related
-
2011
- 2011-01-18 US US13/008,468 patent/US20110151449A1/en not_active Abandoned
- 2011-01-18 US US13/008,182 patent/US9012144B2/en not_active Expired - Fee Related
- 2011-01-18 US US13/008,130 patent/US20110245086A1/en not_active Abandoned
-
2015
- 2015-03-19 US US14/663,010 patent/US9657344B2/en not_active Expired - Fee Related
Patent Citations (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5502773A (en) * | 1991-09-20 | 1996-03-26 | Vanderbilt University | Method and apparatus for automated processing of DNA sequence data |
WO1993021340A1 (en) * | 1992-04-22 | 1993-10-28 | Medical Research Council | Dna sequencing method |
US6087095A (en) * | 1992-04-22 | 2000-07-11 | Medical Research Council | DNA sequencing method |
WO1996027025A1 (en) * | 1995-02-27 | 1996-09-06 | Ely Michael Rabani | Device, compounds, algorithms, and methods of molecular characterization and manipulation with molecular parallelism |
US20020025529A1 (en) * | 1999-06-28 | 2002-02-28 | Stephen Quake | Methods and apparatus for analyzing polynucleotide sequences |
US6818395B1 (en) * | 1999-06-28 | 2004-11-16 | California Institute Of Technology | Methods and apparatus for analyzing polynucleotide sequences |
US20020164629A1 (en) * | 2001-03-12 | 2002-11-07 | California Institute Of Technology | Methods and apparatus for analyzing polynucleotide sequences by asynchronous base extension |
US20090005259A1 (en) * | 2003-02-26 | 2009-01-01 | Complete Genomics, Inc. | Random array DNA analysis by hybridization |
US20050170367A1 (en) * | 2003-06-10 | 2005-08-04 | Quake Stephen R. | Fluorescently labeled nucleoside triphosphates and analogs thereof for sequencing nucleic acids |
US7169560B2 (en) * | 2003-11-12 | 2007-01-30 | Helicos Biosciences Corporation | Short cycle methods for sequencing polynucleotides |
US7491498B2 (en) * | 2003-11-12 | 2009-02-17 | Helicos Biosciences Corporation | Short cycle methods for sequencing polynucleotides |
US7897345B2 (en) * | 2003-11-12 | 2011-03-01 | Helicos Biosciences Corporation | Short cycle methods for sequencing polynucleotides |
Non-Patent Citations (2)
Title |
---|
Tokunaga et al. Single Molecule Imaging of Fluorophores and Enzymatic Reactions Achieved by Objective-Type Total Internal Reflection Fluorescence Microscopy. Biochemical and Biophysical Research Communications 235 : 47 (1997). * |
Tokunaga et al., Single Molecule Imaging of Fluorophores and Enzymatic Reactions Achieved by Objective-Type Total Internal Reflection Fluorescence Microscopy. BBRC 235 : 47 (1997). * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9012144B2 (en) | 2003-11-12 | 2015-04-21 | Fluidigm Corporation | Short cycle methods for sequencing polynucleotides |
US9657344B2 (en) | 2003-11-12 | 2017-05-23 | Fluidigm Corporation | Short cycle methods for sequencing polynucleotides |
US20070099212A1 (en) * | 2005-07-28 | 2007-05-03 | Timothy Harris | Consecutive base single molecule sequencing |
Also Published As
Publication number | Publication date |
---|---|
US9012144B2 (en) | 2015-04-21 |
US20110245086A1 (en) | 2011-10-06 |
US9657344B2 (en) | 2017-05-23 |
US20150292008A1 (en) | 2015-10-15 |
US7491498B2 (en) | 2009-02-17 |
US7897345B2 (en) | 2011-03-01 |
US20050100932A1 (en) | 2005-05-12 |
US20070122828A1 (en) | 2007-05-31 |
US7169560B2 (en) | 2007-01-30 |
US20090191565A1 (en) | 2009-07-30 |
EP1692312A2 (en) | 2006-08-23 |
US20110152114A1 (en) | 2011-06-23 |
EP1692312A4 (en) | 2007-10-17 |
WO2005047523A3 (en) | 2005-12-22 |
WO2005047523A2 (en) | 2005-05-26 |
CA2545619A1 (en) | 2005-05-26 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US9657344B2 (en) | Short cycle methods for sequencing polynucleotides | |
US11643684B2 (en) | Conformational probes and methods for sequencing nucleic acids | |
US20080287306A1 (en) | Methods and devices for sequencing nucleic acids | |
JP2006517798A (en) | Methods and means for nucleic acid sequences | |
US20210071245A1 (en) | Dna amplification technology | |
GB2398301A (en) | A DNA molecule consisting of a stem portion and first and second loop portions | |
US20070031875A1 (en) | Signal pattern compositions and methods | |
US20220010370A1 (en) | Method for sequencing polynucleotides | |
US10669574B2 (en) | DNA amplification technology | |
WO2023154712A1 (en) | Methods, compositions, and systems for long read single molecule sequencing | |
US20100190151A1 (en) | Fluorescently labeled nucleoside triphosphates and analogs thereof for sequencing nucleic acids | |
WO2022271701A2 (en) | Methods and compositions for nucleic acid sequencing |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: BROWN RUDNICK LLP, MASSACHUSETTS Free format text: NOTICE OF ATTORNEY'S LIEN;ASSIGNOR:HELICOS BIOSCIENCES CORPORATION;REEL/FRAME:028060/0898 Effective date: 20120417 |
|
AS | Assignment |
Owner name: ILLUMINA, INC., CALIFORNIA Free format text: LICENSE;ASSIGNOR:FLUIDIGM CORPORATION;REEL/FRAME:030714/0783 Effective date: 20130628 Owner name: COMPLETE GENOMICS, INC., CALIFORNIA Free format text: LICENSE;ASSIGNOR:FLUIDIGM CORPORATION;REEL/FRAME:030714/0686 Effective date: 20130628 Owner name: FLUIDIGM CORPORATION, CALIFORNIA Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:HELICOS BIOSCIENCES CORPORATION;REEL/FRAME:030714/0546 Effective date: 20130628 Owner name: LIFE TECHNOLOGIES CORPORATION, CALIFORNIA Free format text: LICENSE;ASSIGNOR:FLUIDIGM CORPORATION;REEL/FRAME:030713/0655 Effective date: 20130628 Owner name: PACIFIC BIOSCIENCES OF CALIFORNIA, INC., CALIFORNI Free format text: LICENSE;ASSIGNOR:FLUIDIGM CORPORATION;REEL/FRAME:030714/0598 Effective date: 20130628 Owner name: SEQLL, LLC, MASSACHUSETTS Free format text: LICENSE;ASSIGNOR:FLUIDIGM CORPORATION;REEL/FRAME:030714/0633 Effective date: 20130628 |
|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |
|
AS | Assignment |
Owner name: HELICOS BIOSCIENCES CORPORATION, MASSACHUSETTS Free format text: TERMINATION AND RELEASE OF NOTICE OF ATTORNEY'S LIEN;ASSIGNOR:BROWN RUDNICK LLP;REEL/FRAME:046988/0735 Effective date: 20180830 |