US20020012933A1 - Method of sequencing a nucleic acid - Google Patents

Method of sequencing a nucleic acid Download PDF

Info

Publication number
US20020012933A1
US20020012933A1 US09/826,141 US82614101A US2002012933A1 US 20020012933 A1 US20020012933 A1 US 20020012933A1 US 82614101 A US82614101 A US 82614101A US 2002012933 A1 US2002012933 A1 US 2002012933A1
Authority
US
United States
Prior art keywords
nucleic acid
substrate
approximately
sequencing
sequence
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
Application number
US09/826,141
Inventor
Jonathan Rothberg
Joel Bader
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
CuraGen Corp
Original Assignee
CuraGen Corp
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by CuraGen Corp filed Critical CuraGen Corp
Priority to US09/826,141 priority Critical patent/US20020012933A1/en
Publication of US20020012933A1 publication Critical patent/US20020012933A1/en
Abandoned legal-status Critical Current

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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • 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
    • 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

Definitions

  • the present invention relates to methods and apparatuses for determining the sequence of a nucleic acid.
  • DNA sequence polymorphisms can include, e.g., insertions, deletions, or substitutions of nucleotides in one sequence relative to a second sequence.
  • An example of a particular DNA sequence polymorphism is 5′-ATCG-3′, relative to the sequence 5′-ATGG-3′. The first nucleotide ‘G’ in the latter sequence has been replaced by the nucleotide ‘C’ in the former sequence.
  • sequence polymorphism is known as a single-nucleotide polymorphism, or SNP, because the sequence difference is due to a change in one nucleotide.
  • a DNA sequence polymorphism analysis is performed by isolating DNA from an individual, manipulating the isolated DNA, e.g., by digesting the DNA with restriction enzymes and/or amplifying a subset of sequences in the isolated DNA. The manipulated DNA is then examined further to determine if a particular sequence is present.
  • electrophoresis A common application of electrophoresis includes agarose or polyacrylamide gel electrophoresis. DNA sequences are inserted, or loaded, on the gels and subjected to an electric field. Because DNA carries a uniform negative charge, DNA will migrate through the gel based on a charge/mass ratio upon application of the electrical field. Smaller DNA molecules will migrate more rapidly through the gel than larger fragments. After electrophoresis has been continued for a sufficient length of time, the DNA molecules in the initial population of DNA sequences will have separated according to their relative sizes.
  • Particular DNA molecules can then be detected using a variety of detection methodologies.
  • particular DNA sequences are identified by the presence of detectable tags, such as radioactive labels, attached to specific DNA molecules.
  • Electrophoretic-based separation analyses can be less desirable for applications in which it is desirable to rapidly, economically, and accurately analyze a large number of nucleic acid samples for particular sequence polymorphisms.
  • electrophoretic-based analysis can require a large amount of input DNA.
  • processing the large number of samples required for electrophoretic-based nucleic acid based analyses can be labor intensive.
  • electrophoresis can be ill-suited for applications such as clinical sequencing, where relatively cost-effective units with high throughput are needed. Thus, the need for non-electrophoretic methods for sequencing is great. For many applications, electrophoreses is used in conjunction with DNA sequence analysis.
  • solid substrate-based nucleic acid analyses Another alternative to electrophoretic-based separation is analysis is solid substrate-based nucleic acid analyses. These methods typically rely upon the use of large numbers of nucleic acid probes affixed to different locations on a solid support.
  • solid supports can include, e.g., glass surfaces, plastic microtiter plates, plastic sheets, thin polymer, semi-conductors.
  • the probes can be, e.g., adsorbed or covalently attached to the support, or can be microencapsulated or otherwise entrapped within a substrate membrane or film.
  • Substrate-based nucleic acid analyses can include applying a sample nucleic acid known or suspected of containing a particular sequence polymorphism to an array of probes attached to the solid substrate.
  • the nucleic acids in the population are allowed to hybridize to complementarty sequences attached to the substrate, if present. Hybridizing nucleic acid sequences are then detected in a detection step.
  • Solid support matrix-based hybridization and sequencing methodologies can require a high sample-DNA concentration and can be hampered by the relatively slow hybridization kinetics of nucleic acid samples with immobilized oligonucleotide probes. Often, only a small amount of template DNA is available, and it can be desirable to have high concentrations of the target nucleic acid sequence. Thus, substrate based detection analyses often include a step in which copies of the target nucleic acid, or a subset of sequences in the target nucleic acid, is amplified. Methods based on the Polymerase Chain Reaction (PCR), e.g., can increase a small number of probes targets by several orders of magnitude in solution. However, PCR can be difficult to incorporate into a solid-phase approach because the amplified DNA is not immobilized onto the surface of the solid support matrix.
  • PCR Polymerase Chain Reaction
  • the invention is based in part on the discovery of a highly sensitive method for determining the sequences of nucleic acids attached to solid substrates, and of novel substrate services for analyzing nucleic acid sequences.
  • the invention includes a substrate for analyzing a nucleic acid.
  • the substrate includes a fiber optic surface onto which has been affixed one or more nucleic acid sequences.
  • the fiber optic surface can be cavitated, e.g., a hemispherical etching of the opening of a fiber optic.
  • the substrate can in addition include a plurality of bundled fiber optic surfaces, where one or more of the surfaces have anchored primers.
  • the invention includes an apparatus for analyzing a nucleic acid sequence.
  • the apparatus can include a perfusion chamber, wherein the chamber includes a nucleic acid substrate, a conduit in communication with the perfusion chamber, an imaging system, e.g., a fiber optic system, in communication with the perfusion chamber; and a data collection system in communication with the imaging system.
  • the substrate can be a planar substrate. In other embodiments, the substrate can be the afore-mentioned fiber optic surface having nucleic acid sequences affixed to its termini.
  • the invention includes a method for sequencing a nucleic acid.
  • the method includes providing one or more or more nucleic acid anchor primers linked to a solid support and a plurality of circular nucleic acid templates.
  • the nucleic acid anchor primer is then annealed to at least one of the single-stranded circular templates to yield a primed anchor primer-circular template complex.
  • the primed anchor primer-circular template complex is then combined with a polymerase to generate multiple copies of the circular nucleic acid template.
  • a sequencing primer is annealed to the circular nucleic acid template to yield a primed sequencing primer-circular nucleic acid template complex.
  • the sequence primer is the extended with a polymerase and a predetermined nucleotide triphosphate to yield a sequencing product and a sequencing reaction byproduct, e.g., inorganic pyrophosphate. If the predetermined nucleotide is incorporated into the primer, the sequencing reaction byproduct is generated and then identified, thereby determining the sequence of the nucleic acid. If desired, a additional predetermined nucleotide triphosphates can be added, e.g., sequentially, and the presence or absence of sequence byproducts associated with each reaction can be determined.
  • the invention includes a method for sequencing a nucleic acid by providing one or more nucleic acid anchor primers linked to a plurality of anchor primers linked to a fiber optic surface substrate, e.g., the solid substrate discussed above.
  • the solid substrate includes two or more anchoring primers separated by approximately 10 ⁇ m to approximately 200 ⁇ m, 50 ⁇ m to approximately 150 ⁇ m, 100 ⁇ m to approximately 150 ⁇ m, or 150 ⁇ m.
  • the solid support matrix can include a plurality of pads that are covalently linked to the solid support.
  • the surface area of the pads can be, e.g., 10 ⁇ m 2 and one or more pads can be separated from one another by a distance ranging from approximately 50 ⁇ m to approximately 150 ⁇ m.
  • the circular nucleic acid template is single-stranded DNA.
  • the circular template can be, e.g., an open-circle nucleic acid or a closed circle nucleic acid.
  • the circular nucleic acid template can be, e.g., genomic DNA or RNA, or a cDNA copy thereof.
  • the circular nucleic acid can be, e.g., 10-10,000 or 10-1000, 10-200, 10-100, 10-50, or 20-40 nucleotides in length.
  • multiple copies of one or more circular nucleic acids in the population are generated by a polymerase chain reaction.
  • the primed circular template is extended by rolling circle amplification (RCA) to yield a single-stranded concatamer of the annealed circular nucleic acid template.
  • the template amplified by rolling circle amplificaion and be further amplified by annealing a reverse primer to the single-stranded concatamer to yield a primed concatamer template and combining the primed concatamer template with a polymerase enzyme to generate multiple copies of the concatamer template.
  • the template can be extended by a combination of PCR and RCA-amplification.
  • sequencing byproduct analyzed is pyrophosphate.
  • a preferred nucleotide triphosphate for use by the polymerase in extending the primed sequencing primer is a dATP analog, e,g., adenosine 5′-phosphosulfate (APS).
  • the pyrophosphate is detected by contacting the sequencing byproduct with ATP sulfurylase under conditions sufficient to form ATP.
  • the ATP can then be detected, e.g., with an enzyme which generates a detectable product upon reaction with ATP.
  • a preferred enzyme for detecting the ATP is luciferase.
  • a wash buffer can be used between addition of various reactants herein.
  • apyrase is used to remove, e.g., unreacted dNTP used to extend the sequencing primer.
  • the wash buffer can optionally include apyrase.
  • reactants and enzymes used herein e.g., the ATP sulfurylase, luciferase, and apyrase, can be attached to the solid surface.
  • the anchor primer sequence can include, e.g. a biotin group, which can link the anchor primer to the solid support via an avidin group attached to the solid support.
  • the anchor primer is conjugated to a biotin-bovine serum albumin (BSA) moiety.
  • BSA biotin-bovine serum albumin
  • the biotin-BSA moiety can be linked to an avidin-biotin group on the solid support.
  • the biotin-BSA moiety on the anchor primer can be linked to a BSA group on the solid support in the presence of silane.
  • the solid support includes at least one optical fiber.
  • FIG. 1 is a schematic illustration of rolling circle based amplification using an anchor primer.
  • FIG. 2 is a drawing of a sequencing apparatus according to the present invention.
  • FIG. 3 is a drawing of a perfusion chamber according to the present invention.
  • FIG. 4 is a drawing of a cavitated fiber optic terminus of the present invention.
  • the methods described herein include a sample preparation process in which multiple copies of individual single-stranded nucleic acid molecules, termed anchor primers, are linked to a solid substrate.
  • anchor primers multiple copies of individual single-stranded nucleic acid molecules
  • a region of the substrate containing at least one linked anchor primer is a anchor pad.
  • a plurality of anchor primers linked on a single solid surface can form an array.
  • a plurality of nucleic acid template sequences is then annealed to the array to form one or more primed circular templates.
  • the primed circular templates are next amplified.
  • a sequencing primer is annealed to the amplified nucleic acid and used to generate a sequencing product.
  • the nucleotide sequence of the sequence product is then determined, thereby allowing for the determination of the nucleic acid.
  • the methods and apparatuses described herein allow for the determination of nucleic acid sequence information without the need for first cloning a nucleic acid.
  • the method is highly sensitive and can be used to determine the nucleotide sequence of a template nucleic acid which is present in only a few copies in a starting population of nucleic acids.
  • the methods and apparatuses described are generally useful for any application which the identification of any particular nucleic acid sequence is desired.
  • the methods allow for identification of single nucleotide polymorphisms (SNPs) and transcript profiling.
  • SNPs single nucleotide polymorphisms
  • Other uses include sequencing of artificial DNA constructs to confirm or elicit their primary sequence, or to isolate specific mutant clones from random mutagenesis screens, as well as to obtain the sequence of cDNA from single cells, whole tissues or organisms from any developmental stage or environmental circumstance in order to determine the gene expression profile from that specimen.
  • the methods allow for the sequencing of PCR products and/or cloned DNA fragments of any size isolated from any source.
  • the methods of the present invention can be also used for the sequencing of DNA fragments generated by analytical techniques that probe higher order DNA structure by their differential sensitivity to enzymes, radiation or chemical treatment (e.g., partial DNase treatment of chromatin), or for the determination of the methylation status of DNA by comparing sequence generated from a given tissue with or without prior treatment with chemicals that convert methyl-cytosine to thymine (or other nucleotide) as the effective base recognized by the polymerase. Further, the methods of the present invention can be used to assay cellular physiology changes occurring during development or senescence at the level of primary sequence.
  • Anchor primers in general include a stalk region and at least two contiguous adapter regions.
  • the stalk region is present at the 5′ end of the anchor primer and includes a region of nucleotides for attaching the anchor primer to the solid substrate.
  • the anchor primer in general includes a region which hybridizes to a complementary sequence present in one or more members of a population of nucleic acid sequences.
  • the anchor primer includes two adjoining regions which hybridize to complementary regions ligated to separate ends of a target nucleic acid sequence. This embodiment is illustrated in FIG. 1, which is discussed in more detail below.
  • the adapter regions in the anchor primers are complementary to non-contiguous regions of sequence present in a second nucleic acid sequence.
  • Each adapter region for example, can be homologous to each terminus of a fragment produced by digestion with one or more restriction endonucleases.
  • the fragment can include, e.g. a sequence known or suspected to contain a sequence polymorphism.
  • the anchor primer may contain two adapter regions that are homologous to a gapped, i.e., non-contiguous because of a deletion of one or more nucleotides, region of a target nucleic acid sequence.
  • a target nucleic acid sequence e.g., a target sequence in population of nucleic acids sequences.
  • an aligning oligonucleotide corresponding to the gapped sequence may be annealed to the anchor primer along with a population of template nucleic acid molecules.
  • the anchor primer may optionally contain additional elements, e.g., one or more restriction enzyme recognition sites, RNA polymerase binding sites (e.g., a T7 promoter site).
  • additional elements e.g., one or more restriction enzyme recognition sites, RNA polymerase binding sites (e.g., a T7 promoter site).
  • One or more of the adapter regions may include, e.g., a restriction enzyme recognition site or sequences present in identified DNA sequences, e.g., sequences present in known genes.
  • One or more adapter regions may also include sequences known to flank sequence polymorphisms.
  • Sequence polymorphisms include nucleotide substitutions, insertions, deletions, or other rearrangements which result in a sequence difference between two otherwise identical nucleic acid sequences.
  • An example of a sequence polymorphism is a single nucleotide polymorphism (SNP).
  • any nucleic acid capable of base-pairing can be used as an anchor primer.
  • the anchor primer is an oligonucleotide.
  • oligonucleotide includes linear oligomers of natural or modified monomers or linkages, e.g., deoxyribonucleosides, ribonucleosides, anomeric forms thereof, peptide nucleic acids (PNAs), and the like, that are capable of specifically binding to a target polynucleotide by way of a regular pattern of monomer-to-monomer interactions.
  • These types of interactions can include, e.g., Watson-Crick type of base-pairing, base stacking, Hoogsteen or reverse-Hoogsteen types of base-pairing, or the like.
  • the monomers are linked by phosphodiester bonds, or analogs thereof, to form oligonucleotides ranging in size from, e.g., 3-200, 8-150, 10-100, 20-80, or 25-50 monomeric units.
  • oligonucleotide is represented by a sequence of letters, it is understood that the nucleotides are oriented in the 5′ ⁇ 3′ direction, from left-to-right, and that the letter “A” donates deoxyadenosine, the letter “T” denotes thymidine, the letter “C” denotes deoxycytosine, and the letter “G” denotes deoxyguanosine, unless otherwise noted herein.
  • the oligonucleotides of the present invention can include non-natural nucleotide analogs. However, where, for example, processing by enzymes is required, or the like, oligonucleotides comprising naturally-occurring nucleotides are generally required for maintenance of biological function.
  • any material can be used as the solid support material, as long as the surface allows for stable attachment of the primers and detection of nucleic acid sequences.
  • the solid support material can be planar or can be cavitated, e.g., in a cavitated terminus of a fiber optic.
  • the solid support is optically transparent, e.g., glass.
  • the anchor primer can be linked to the solid support to reside on or within the solid support.
  • the plurality of anchor primers is linked to the solid support so they are spaced regular intervals within an array.
  • the periodicity between primers is preferably greater than either the diffusion rate of the products of the sequencing reactions or the optical resolving power of the detection system, both of which are described in more detail below.
  • the distance between primers on a solid substrate can be, 10-400 ⁇ m, 50-150 ⁇ m, 100-150 ⁇ m, or 150 ⁇ m.
  • An array of attachment sites on the optically transparent solid support is constructed using lithographic techniques commonly used in the construction of electronic integrated circuits as described in, e.g., techniques for attachment described in U.S. Pat. Nos. 5,5143,854, 5,445,934, 5,744,305, and 5, 800,992; Chee et al., Science 274: 610-614 (1996); Fodor et al., Nature 364: 555-556 (1993); Fodor et al., Science 251: 767-773 (1991); Gushin, et al., Anal. Biochem.
  • Photolithography and electron beam lithography sensitize the solid support or substrate with a linking group that allows attachment of a modified biomolecule (e.g., proteins or nucleic acids).
  • a modified biomolecule e.g., proteins or nucleic acids
  • Anchor primers are linked to the solid substrate at the sensitized sites.
  • a region of a solid substrate containing a linked primer is an anchor pad.
  • the anchor pads can, e.g., small diameter spots etched at evenly spaced intervals on the solid support.
  • the anchor primer can be attached to the solid support via a covalent or non-covalent interaction.
  • linkages common in the art include Ni 2+ /hexahistidine, streptavidin/biotin, avidin/biotin, glutathione S-transferase (GST)/glutathione, monoclonal antibody/antigen, and maltose binding protein/maltose. Samples containing the appropriate tag are incubated with the sensitized substrate so that a single molecule attaches at each sensitized site.
  • biotin-(strept-)avidin methodology provides several different ways to immobilize the anchor on the solid support.
  • One biotin-(strept-)avidin-based anchoring method uses a thin layer of a photoactivatable biotin analog dried onto a solid surface. (Hengsakul and Cass, 1996. Biocongjugate Chem. 7: 249-254). The biotin analog is then exposed to white light through a mask, so as to create defined areas of activated biotin. Avidin (or streptavidin) is then added and allowed to bind to the activated biotin. The avidin possesses free biotin binding sites which can be utilized to “anchor” the biotinylated oligonucleotides through a biotin-(strept-)avidin linkage.
  • the anchor primer can be attached to the solid support with a biotin derivative possessing a photo-removable protecting group.
  • This moiety is covalently bound to bovine serum albumin (BSA), which is attached to the solid support, e.g., a glass surface.
  • BSA bovine serum albumin
  • a mask is then used to create activated biotin within the defined irradiated areas.
  • Avidin may then be localized to the irradiated area, with biotinylated DNA subsequently attached through a BSA-biotin-avidin-biotin link.
  • an intermediate layer of silane is deposited in a self-assembled monolayer on a silicon dioxide silane surface that can be patterned to localize BSA binding in defined regions. See e.g., Mooney, et al., 1996. Proc. Natl. Acad. Sci. USA 93: 12287-12291.
  • each sensitized site on a solid support is potentially capable of attaching multiple anchor primers.
  • each anchor pad may include one or more anchor primers. It is preferable to maximize the number of pads that have only a single productive reaction center (e.g., the number of pads that, after the extension reaction, have only a single sequence extended from the anchor primer).
  • each individual pad contains just one linked anchor primer.
  • Pads having only one anchor primer can be made by performing limiting dilutions of a selected anchor primer on to the solid support such that, on average, only one anchor primer is deposited on each pad.
  • the concentration of anchor primer to be applied to a pad can be calculated utilizing, for example, a Poisson distribution model.
  • a range of anchor primer concentrations and circular template concentrations may be subsequently scanned to find a value of N p f closest to 1.
  • a preferable method to optimize this distribution is to allow multiple anchor primers on each reaction pad, but use a limiting dilution of circular template so that, on average, only one primer on each pad is extended to generate the sequencing template.
  • anchor primers will likely be bound on each reaction pad, but a limiting dilution of circular template may be used so that, on average, only one primer on each pad anneals to a template molecule and is extended to amplify the sequencing template.
  • the individual pads are approximately 10 ⁇ m on a side, with a 100 ⁇ m spacing between adjacent pads.
  • a total of approximately 10,000 pads could be deposited, and, according to the Poisson distribution, approximately 3700 of these will contain a single anchor primer.
  • modified, e.g., biotinylated enzymes are deposited to bind to the remaining, unused avidin binding sites on the planar surface.
  • multiple anchor primers are attached to any one individual pad in an array.
  • Limiting dilutions of a plurality of circular nucleic acid templates may be hybridized to the anchor primers so immobilized such that, on average, only one primer on each pad is hybridized to a nucleic acid template.
  • Library concentrations to be used may be calculated utilizing, for example, limiting dilutions and a Poisson distribution model.
  • a plurality of nucleic acid templates in general includes open circular or closed circular nucleic acid molecules.
  • a “closed circle” is a covalently closed circular nucleic acid molecule, e.g., a circular DNA or RNA molecule.
  • An “open circle” is a linear single-stranded nucleic acid molecule having a 5′ phosphate group and a 3′ hydroxyl group. The ends of a given open circle nucleic acid molecule can be ligated by DNA ligase.
  • Sequences at the 5′ and 3′ ends of the open circle molecule are complementary to two regions of adjacent nucleotides in a second nucleic acid molecule, e.g., an adapter region of an anchor primer, or to two regions that are nearly adjoining in a second DNA molecule.
  • the ends of the open-circle molecule can be ligated using DNA ligase, or extended by DNA polymerase in a gap-filling reaction.
  • Open circles are described in detail in Lizardi, U.S. Pat. No. 5,854,033.
  • An open circle can be converted to a closed circle in the presence of a DNA ligase (for DNA) or RNA ligase following, e.g., annealing of the open circle to an anchor primer.
  • Circularized oligonucleotide probes are comprised of two target sequence-complementarity sequences which are connected by a linker which may possess detectable functionalities.
  • the linkers can be ligated to ends of members of a library of nucleic acid sequences that have been, e.g., physically sheared or digested with restriction endonucleases.
  • the 5′- and 3′-terminal regions of these linear oligonucleotides are designed to basepair adjacent to one another on a specific target sequence strand, thus the termini of the linear oligonucleotide are brought into juxtaposition by hybridization to the target sequence.
  • This juxtaposition allows the two probe segments (if properly hybridized) to be covalently-bound by enzymatic ligation (e.g., with T 4 DNA ligase), thus converting the probes to circularly-closed molecules which are catenated to the specific target sequences (see e.g., Nilsson, et al., 1994. Science 265: 2085-2088).
  • the resulting probes are suitable for the simultaneous analysis of many gene sequences both due to their specificity and selectivity for gene sequence variants (see e.g., Lizardi, et al., 1998. Nat. Genet. 19: 225-232; Nilsson, et al., 1997. Nat. Genet. 16: 252-255) and due to the fact that the resulting reaction products remain localized to the specific target sequences. Moreover, intramolecular ligation of many different probes is expected to be less susceptible to non-specific cross-reactivity than multiplex PCR-based methodologies where non-cognate pairs of primers can give rise to irrelevant amplification products (see e.g., Landegren and Nilsson, 1997. Ann. Med. 29: 585-590).
  • the starting library can be either single-stranded or double-stranded, as long as it includes a region that, if present in the library, is available for annealing, or can be made available for annealing, to an anchor primer sequence.
  • Library templates can include multiple elements, including, but not limited to, one or more regions that are complementary to the anchor primer.
  • the template libraries may include a region complementary to a sequencing primer, a control nucleotide region, and an insert sequence comprised of the sequencing template to be subsequently characterized.
  • the control nucleotide region is used to calibrate the relationship between the amount of byproduct and the number of nucleotides incorporated.
  • the term “complement” refers to nucleotide sequences that are able to hybridize to a specific nucleotide sequence to form a matched duplex.
  • a library template includes: (i) two distinct regions that are complementary to the anchor primer, (ii) one region complementary to the sequencing primer, (iii) one control nucleotide region, (iv) an insert sequence of 30-100 nucleotides that is to be sequenced.
  • the template can, of course, include two, three, or all four of these features.
  • the template nucleic acid can be constructed from any source of nucleic acid, e.g., any cell, tissue, or organism, and can be generated by any art-recognized method. Suitable methods include, e.g., sonication of genomic DNA and digestion with one or more restriction endonucleases (RE) to fragment a population of nuclei acid molecules, e.g., genomic DNA.
  • RE restriction endonucleases
  • one or more of the restriction enzymes have distinct four-base recognition sequences. Examples of such enzymes include, e.g., Sau3AI, MspI, and TaqI.
  • the enzymes are used in conjunction with anchor primers having regions containing recognition sequences for the corresponding restriction enzymes.
  • the one or both adapter regions anchor primers contain additional sequences adjoining known restriction enzyme recognition sequences, thereby allowing for capture or annealing of specific restriction fragments of interest to the anchor primer.
  • the restriction enzyme is used with a type IIS restriction enzyme.
  • template libraries can be made by generating a complementary DNA (cDNA) library from RNA, e.g., messenger RNA (mRNA).
  • cDNA complementary DNA
  • the cDNA library can, if desired, be further processed with restriction endonucleases to obtain either 3′ signature sequences, internal fragments, or 5′ fragments.
  • a number of in vitro nucleic acid amplification techniques may be utilized to extend the anchor primer sequence.
  • the size of the amplified DNA should be smaller than the size of the anchor pad and also smaller than the distance between anchoring pads.
  • the amplification is typically performed in the presence of a polymerase, e.g., a DNA or RNA-directed DNA polymerase, and one, two, three, or four types of nucleotide triphosphates, and, optionally, auxiliary binding proteins.
  • a polymerase e.g., a DNA or RNA-directed DNA polymerase
  • any polymerase capable of extending a primed 3′-OH group can be used a long as it lacks a 3′ to 5′ exonuclease activity.
  • Suitable polymerases include, e.g., the DNA polymerases from Bacillus stearothermophilus, Thermus acquaticus, Pyrococcus furiosis, Thermococcus litoralis, and Thermus thermophilus, bacteriophage T 4 and T 7 , and the E. coli DNA polymerase I Klenow fragment.
  • Suitable RNA-directed DNA polymerases include, e.g., the reverse transcriptase from the Avian Myeloblastosis Virus, the reverse transcriptase from the Moloney Murine Leukemia Virus, and the reverse transcriptase from the Human Immunodeficiency Virus-I.
  • PCR polymerase chain reaction
  • ligase chain reaction see e.g., Barany, 1991. Proc. Natl. Acad. Sci. USA 88: 189-193; Barringer, et al., 1990. Gene 89: 117-122
  • transcription-based amplification see e.g., Kwoh, et al., 1989. Proc. Natl. Acad. Sci.
  • Isothermal amplificaion also includes rolling circle-based amplification (RCA).
  • RCA is discussed in, e.g., Kool, U.S. Pat. No. 5,714,320 and Lizardi, U.S. Pat. No. 5,854,033; Hatch, et al., 1999. Genet. Anal. Biomol. Engineer. 15: 35-40.
  • the result of the RCA is a single DNA strand extended from the 3′ terminus of the anchor primer (and thus is linked to the solid support matrix) and including a concatamer containing multiple copies of the circular template annealed to a primer sequence.
  • 10,000 or more copies of circular templates each having a size of approximately 100 nucleotides size range, can be obtained with RCA.
  • FIG. 1 The product of RCA amplification following annealing of a circular nucleic acid molecule to an anchor primer is shown schematically in FIG. 1.
  • a circular template nucleic acid 102 is annealed to an anchor primer 104 , which has been linked to a surface 106 at its 5′ end and has a free 3′ OH available for extension.
  • the circular template nucleic acid 102 includes two adapter regions 108 and 110 which are homologous to regions of sequence in the anchor primer 104 .
  • an insert 112 and a region 114 homologous to a sequencing primer which is used in the sequencing reactions described below.
  • the free 3′-OH on the anchor primer 104 can be extended using sequences within the template nucleic acid 102 .
  • the anchor primer 102 can be extended along the template multiple times, with each iteration adding to the sequence extended from the anchor primer a sequence complementary to the circular template nucleic acid.
  • Four iterations, or four rounds of rolling circle replication, are shown in FIG. 1 as the extended anchor primer amplification product 114 .
  • Extension of the anchor primer results in an amplification product covalently attached to the substrate 106 .
  • Circular oligonucleotides which are generated during polymerase-mediated DNA replication are dependent upon the relationship between the template and the site of replication initiation.
  • the critical features include whether the template is linear or circular in nature, and whether the site of initiation of replication (i.e., the replication “fork”) is engaged in synthesizing both strands of DNA or only one.
  • the replication fork is treated as the site at which the new strands of DNA are synthesized.
  • linear molecules whether replicated unidirectionally or bidirectionally
  • the movement of the replication fork(s) generate a specific type of structural motif. If the template is circular, one possible spatial orientation of the replicating molecule takes the form of an ⁇ structure.
  • RCA can occur when the replication of the duplex molecule begins at the origin. Subsequently, a nick opens one of the strands, and the free 3′-terminal hydroxyl moiety generated by the nick is extended by the action of DNA polymerase. The newly synthesized strand eventually displaces the original parental DNA strand.
  • This aforementioned type of replication is known as rolling-circle replication (RCR) because the point of replication may be envisaged as “rolling around” the circular template strand and, theoretically, it could continue to do so indefinitely. As it progresses, the replication fork extends the outer DNA strand the previous partner.
  • the displaced strand possesses the original genomic sequence (e.g., gene or other sequence of interest) at its 5′-terminus.
  • the original genomic sequence is followed by any number of “replication units” complementary to the original template sequence, wherein each replication unit is synthesized by continuing revolutions of said original template sequence. Hence, each subsequent revolution displaces the DNA which is synthesized in the previous replication cycle.
  • rolling-circle replication is utilized in several biological systems.
  • their genome consists of single-stranded, circular DNA.
  • the circular DNA is initially converted to a duplex form, which is then replicated by the aforementioned rolling-circle replication mechanism.
  • the displaced terminus generates a series of genomic units, which can be cleaved and inserted into the phage particles, or they can be utilized for further replication cycles by the phage.
  • the displaced single-strand of a rolling-circle can be converted to duplex DNA by synthesis of a complementary DNA strand. This synthesis can be used to generate the concatemeric duplex molecules required for the maturation of certain phage DNAs.
  • this provides the principle pathway by which ⁇ bacteriophage matures.
  • Rolling-circle replication is also used in vivo to generate amplified rDNA in Xenopus oocytes, and this fact may help explain why the amplified rDNA is comprised of a large number of identical repeating units.
  • a single genomic repeating unit is converted into a rolling-circle.
  • the displaced terminus is then converted into duplex DNA which is subsequently cleaved from the circle so that the two termini can be ligated together so as to generate the amplified circle of rDNA.
  • a strand may be generated which represents many tandem copies of the complement to the circularized molecule.
  • RCR has recently been utilized to obtain an isothermal cascade amplification reaction of circularized padlock probes in vitro in order to detect single-copy genes in human genomic DNA samples (see Lizardi, et al., 1998. Nat. Genet. 19: 225-232).
  • RCR has also been utilized to detect single DNA molecules in a solid phase-based assay, although difficulties arose when this technique was applied to in situ hybridization (see Lizardi, et al., 1998. Nat. Genet. 19: 225-232).
  • Rolling circle DNA synthesis small circular oligonucleotides as efficient templates for DNA polymerase. J. Am. Chem. Soc. 118: 1587-1594; Fire and Xu, 1995. Rolling replication of short DNA circles. Proc. Natl. Acad. Sci. USA 92: 4641-4645; Nilsson, et al., 1994. Padlock probes: circularizing oligonucleotides for localized DNA detection. Science 265: 2085-2088). RCA targets specific DNA sequences through hybridization and a DNA ligase reaction. The circular product is then subsequently used as a template in a rolling circle replication reaction.
  • Rolling-circle amplification (RCA) driven by DNA polymerase can replicate circularized oligonucleotide probes with either linear or geometric kinetics under isothermal conditions.
  • a complex pattern of DNA strand displacement ensues which possesses the ability to generate 1 ⁇ 10 9 or more copies of each circle in a short period of time (i.e., less-than 90 minutes), enabling the detection of single-point mutations within the human genome.
  • RCA uses a single primer, RCA generates hundreds of randomly-linked copies of a covalently closed circle in several minutes.
  • the DNA product remains bound at the site of synthesis, where it may be labeled, condensed and imaged as a point light source.
  • linear oligonucleotide probes which can generate RCA signals, have been bound covalently onto a glass surface. The color of the signal generated by these probes indicates the allele status of the target, depending upon the outcome of specific, target-directed ligation events.
  • RCA permits millions of individual probe molecules to be counted and sorted, it is particularly amenable for the analysis of rare somatic mutations. RCA also shows promise for the detection of padlock probes bound to single-copy genes in cytological preparations.
  • a solid-phase RCA methodology has also been developed to provide an effective method of detecting constituents within a solution. Initially, a recognition step is used to generate a complex consisting of a DNA primer duplexed with a circular template is bound to a surface. A polymerase enzyme is then used to amplify the bound complex. RCA uses small DNA probes that are amplified to provide an intense signal using detection methods, including the methods described in more detail below.
  • isothermal amplification systems include, e.g., (i) self-sustaining, sequence replication (see e.g., Guatelli, et al., 1990. Proc. Natl. Acad. Sci USA 87: 1874-1878), (ii) the Q ⁇ replicase system (see e.g., Lizardi, et al., 1988. BioTechnology 6: 1197-1202), and (iii) nucleic acid sequence-based amplification (NASBATM; see Kievits, et al., 1991. J. Virol. Methods 35: 273-286).
  • sequence replication see e.g., Guatelli, et al., 1990. Proc. Natl. Acad. Sci USA 87: 1874-1878
  • Q ⁇ replicase system see e.g., Lizardi, et al., 1988. BioTechnology 6: 1197-1202
  • NASBATM nucleic acid sequence-based amplification
  • Amplification of a nucleic acid template as described above results in multiple copies of a template nucleic acid sequence covalently linked to an anchor primer.
  • a region of the sequence product is determined by annealing a sequencing primer to region of the template nucleic acid, and then contacting the sequencing primer with a DNA polymerase and a known nucleotide triphosphate, i.e., dATP, dCTP, dGTP, dTTP, or an analog of one of these nucleotides.
  • the sequence primer can be any length or base composition, as long as it is capable of specifically annealing to a region of the amplified nucleic acid template. No particular structure is required for the sequencing primer is required so long as it is able to specifically prime a region on the amplified template nucleic acid.
  • the sequencing primer is complementary to a region of the template that is between the sequence to be characterized and the sequence hybridizable to the anchor primer.
  • the sequencing primer is extended with the DNA polymerase to form a sequence product. The extension is performed in the presence of one or more types of nucleotide triphosphates, and if desired, auxiliary binding proteins.
  • Incorporation of the dNTP is determined by assaying for the presence of a sequencing byproduct.
  • the nucleotide sequence of the sequencing product is determined by measuring inorganic pyrophosphate (PPi) liberated from a nucleotide triphosphate (dNTP) as the NTP is incorporated into an extended sequence primer.
  • PPi inorganic pyrophosphate
  • dNTP nucleotide triphosphate
  • This method of sequencing termed PyrosequencingTM technology (PyroSequencing AB, Sweden) can be performed in solution (liquid phase) or as a solid phase technique.
  • PPi-based sequencing methods are described generally in, e.g., WO9813523A1, Ronaghi, et al., 1996. Anal. Biochem. 242: 84-89, and Ronaghi, et al., 1998. Science 281: 363-365 (1998). These disclosures of PPi sequencing are incorporated herein in their entirety, by
  • Pyrophosphate released under these conditions can be detected enzymatically (e.g., by the generation of light in the luciferase-luciferin reaction). Such methods enable a nucleotide to be identified in a given target position, and the DNA to be sequenced simply and rapidly while avoiding the need for electrophoresis and the use of potentially dangerous radiolabels.
  • PPi can be detected by a number of different methodologies, and various enzymatic methods have been previously described (see e.g., Reeves, et al., 1969. Anal. Biochem. 28: 282-287; Guillory, et al., 1971. Anal. Biochem. 39: 170-180; Johnson, et al., 1968. Anal. Biochem. 15: 273; Cook, et al., 1978. Anal. Biochem. 91: 557-565; and Drake, et al, 1979. Anal. Biochem. 94: 117-120).
  • PPi liberated as a result of incorporation of a dNTP by a polymerase can be converted to ATP using, e.g., an ATP suflufurylase.
  • This enzyme has been identified as being involved in sulfur metabolism. Sulfur, in both reduced and oxidized forms, is an essential mineral nutrient for plant and animal growth (see e.g., Schmidt and Jager, 1992. Ann. Rev. Plant Physiol. Plant Mol. Biol. 43: 325-349). In both plants and microorganisms, active uptake of sulfate is followed by reduction to sulfide.
  • ATP sulfurylase has been highly purified from several sources, such as Saccharomyces cerevisiae (see e.g., Hawes and Nicholas, 1973. Biochem. J. 133: 541-550); Penicillium chrysogenum (see e.g., Renosto, et al., 1990. J. Biol. Chem. 265: 10300-10308); rat liver (see e.g., Yu, et al., 1989. Arch. Biochem. Biophys. 269: 165-174); and plants (see e.g., Shaw and Anderson, 1972. Biochem. J. 127: 237-247; Osslund, et al., 1982. Plant Physiol.
  • ATP sulfurylase genes have been cloned from prokaryotes (see e.g., Leyh, et al., 1992. J. Biol. Chem. 267: 10405-10410; Schwedock and Long, 1989. Mol. Plant Microbe Interaction 2: 181-194; Laue and Nelson, 1994. J. Bacteriol. 176: 3723-3729); eukaryotes (see e.g., Cherest, et al., 1987. Mol. Gen. Genet. 210: 307-313; Mountain and Korch, 1991. Yeast 7: 873-880; Foster, et al., 1994. J. Biol. Chem.
  • the enzyme is homo-oligomer or heterodimer, depending upon the specific source (see e.g., Leyh and Suo, 1992. J. Biol Chem. 267: 542-545).
  • ATP sulfurylase has been used for many different applications, for example, bioluminometric detection of ADP at high concentrations of ATP (see e.g., Schultz, et al., 1993. Anal. Biochem. 215: 302-304); continuous monitoring of DNA polymerase activity (see e.g., Nyrbn, 1987. Anal. Biochem. 167: 235-238); and DNA sequencing (see e.g., Ronaghi, et al., 1996. Anal. Biochem. 242: 84-89; Ronaghi, et al., 1998. Science 281: 363-365; Ronaghi, et al., 1998. Anal. Biochem. 267: 65-71).
  • the colorimetric molybdolysis assay is based on phosphate detection (see e.g., Wilson and Bandurski, 1958. J. Biol. Chem. 233: 975-981), whereas the continuous spectrophotometric molybdolysis assay is based upon the detection of NADH oxidation (see e.g., Seubert, et al., 1983. Arch. Biochem. Biophys. 225: 679-691; Seubert, et al., 1985. Arch. Biochem. Biophys. 240: 509-523).
  • the later assay requires the presence of several detection enzymes.
  • radioactive assays have also been described in the literature (see e.g., Daley, et al., 1986. Anal. Biochem. 157: 385-395).
  • one assay is based upon the detection of 32 PPi released from 32 P-labeled ATP (see e.g., Seubert, et al., 1985. Arch. Biochem. Biophys. 240: 509-523) and another on the incorporation of 35 S into [ 35 S]-labeled APS (this assay also requires purified APS kinase as a coupling enzyme; see e.g., Seubert, et al., 1983. Arch. Biochem. Biophys.
  • ATP produced by an ATP sulfurylase can be converted using enzymatic reactions which convert ATP to light.
  • Light-emitting chemical reactions i.e., chemiluminescence
  • biological reactions i.e., bioluminescence
  • bioluminescent reactions the chemical reaction that leads to the emission of light is enzyme-catalyzed.
  • the luciferin-luciferase system allows for specific assay of ATP and the bacterial luciferase-oxidoreductase system can be used for monitoring of NAD(P)H.
  • Suitable enzymes for converting ATP into light include luciferases, e.g., insect luciferases. Luciferases produce light as an end-product of catalysis.
  • the best known light-emitting enzyme is that of the firefly, Photinus pyralis (Coleoptera).
  • the corresponding gene has been cloned and expressed in bacteria (see e.g., de Wet, et al., 1985. Proc. Natl. Acad. Sci. USA 80: 7870-7873) and plants (see e.g., Ow, et al., 1986. Science 234: 856-859), as well as in insect (see e.g., Jha, et al., 1990.
  • Firefly luciferase catalyzes bioluminescence in the presence of luciferin, adenosine 5′-triphosphate (ATP), magnesium ions, and oxygen, resulting in a quantum yield of 0.88 (see e.g., McElroy and Selinger, 1960. Arch. Biochem. Biophys. 88: 136-145).
  • the firefly luciferase bioluminescent reaction can be utilized as an assay for the detection of ATP with a detection limit of approximately 1 ⁇ 10 ⁇ 13 M (see e.g., Leach, 1981. J. Appl. Biochem. 3: 473-517).
  • the sequence primer is exposed to a polymerase and a known dNTP. If the dNTP is incorporated onto the 3′ end of the primer sequence, the dNTP is cleaved and a PPi molecule is liberated. The PPi is then converted to ATP with ATP sulfurylase.
  • the ATP sulfurylase is present at a sufficiently high concentration that the conversion of PPi proceeds with first-order kinetics. In the presence of luciferase, the ATP is hydrolyzed to liberate a photon.
  • the reaction preferably has a sufficient concentration of luciferase present within the reaction mixture such that the reaction, ATP ⁇ ADP+PO 4 3 ⁇ + photon (light), proceeds with first-order kinetics.
  • the photon can be measured using methods and apparatuses described below.
  • wash buffer any wash buffer used in pyrophosphate sequencing can be used.
  • An example of a wash buffer is 10 mM Trisc-HCl (pH 7.5), 1 mM EDTA, 2 M NaCl, 1% Tween 20 (Nyren et al., Anal. Biochem. 208:171-75, 1993).
  • the concentration of reactants in the sequencing reaction include 1 pmol DNA, 3 pmol polymerase, 40 pmol dNTP in 0.2 ml buffer. See Ronaghi, et al., Anal. Biochem. 242: 84-89 (1996).
  • the sequencing reaction can be performed with each of four predetermined nucleotides, if desired.
  • a “complete” cycle generally includes sequentially administering sequencing reagents for each of the nucleotides dATP, dGTP, dCTP and dTTP (or dUTP), in a predetermined order. Unincorporated dNTPs are washed away between each of the nucleotide additions. Alternatively, unincorporated dNTPs are degraded by apyrase (see below). The cycle is repeated as desired until the desired amount of sequence of the sequence product is obtained. In some embodiments, about 10-1000, 10-100, 10-75, 20-50, or about 30 nucleotides of sequence information is obtained from one annealed primer.
  • Luciferase can hydrolyze dATP directly with concomitant release of a photon. This results in a false positive signal because the hydrolysis occurs independent of incorporation of the dATP into the extended sequencing primer.
  • a dATP analog can be used which is incorporated into DNA, i e., it is a substrate for a DNA polymerase, but is not a substrate for luciferase.
  • One such analog is ⁇ -thio-ATP such as adenosine 5′-phosphosulfate (APS).
  • APS adenosine 5′-phosphosulfate
  • the PPi-based detection is calibrated by the measurement of the light released following the addition of control nucleotides to the sequencing reaction mixture immediately after the addition of the sequencing primer. This allows for normalization of the reaction conditions. Incorporation of two or more identical nucleotides in succession is revealed by a corresponding increase in the amount of light released. Thus, a two-fold increase in released light relative to control nucleotides reveals the incorporation of two successive dNTPs into the extended primer.
  • apyrase may be “washed” or “flowed” over the surface of the solid support so as to facilitate the degradation of any remaining, non-incorporated dNTPs within the sequencing reaction mixture. Upon treatment with apyrase, any remaining reactants are washed away in preparation for the following dNTP incubation and photon detection steps. Alternatively, the apyrase may be bound to the solid support.
  • the pyrophosphate sequencing reactions preferably take place in a thin, aqueous reaction chamber comprising an optically-transparent solid support surface and an optically-transparent cover. Sequencing reagents may then be delivered by flowing them across the surface of the substrate. When the support is not planar, the reagents may be delivered by dipping the solid support into baths of any given reagents.
  • the enzymes utilized in the pyrophosphate sequencing reaction may be immobilized onto the solid support.
  • luciferase is immobilized, it is preferably less than 50 ⁇ m from an anchored primer.
  • the photons generated by luciferase may be quantitated using a variety of detection apparatuses, e.g., a photomultiplier tube, charge-coupled display (CCD), absorbance photometer, and a luminometer, as well as the apparatuses described herein.
  • the quantitation of the emitted photons is accomplished by the use of charge-coupled display (CCD) camera fitted with a microchannel plate intensifier.
  • CCD detectors are described in, e.g., Bronks, et al., 1995. Anal Chem. 65: 2750-2757.
  • the CCD camera uses a custom designed and fabricated CCD possessing a total of 16 million pixels (i.e., 4,000 ⁇ 4,000 pixel array) which can detect approximately 1% of the photons produced and can convert 40% to 80% of the photons produced into an actual measurable signal. With this system, approximately 1% of the photons produced are detected. This system can convert 40% to 80% of the photons produced into an actual measurable signal. Additionally, this CCD system possesses a minimum signal-to-noise ratio of 5:1, with a 10:1 signal-to-noise ratio being preferable.
  • apparatuses for sequencing nucleic acids include anchor primers attached to planar substrates. Nucleic acid sequence information can be detected using conventional optics or fiber-optic based systems attached to the planar substrate. In other embodiments, the apparatuses include anchor primers attached to the termini of fiber-optic arrays. In these embodiments, sequence information can be obtained directly from the termini of the fiber optic array.
  • FIG. 2 An apparatus for sequencing nucleic acids is illustrated in FIG. 2.
  • the apparatus includes an inlet conduit 200 in communication with a detachable perfusion chamber 220 .
  • the inlet conduit 200 allows for entry of sequencing reagents via a plurality of tubes 202 - 212 , which are each in communication with a plurality of sequencing dispensing reagent vessels 214 - 224 .
  • Reagents are introduced through the conduit 200 into the perfusion chamber 220 using either a pressurized system or pumps to drive positive flow.
  • the reagent flow rates are from 1 to 50 ml/minute with volumes from 0.100 ml to continuous flow (for washing).
  • Valves are under computer control to allow cycling of nucleotides and wash reagents.
  • Sequencing reagents, e.g., polymerase can be either pre-mixed with nucleotides or added in stream.
  • a manifold brings all six tubes 202 - 212 together into one for feeding the perfusion chamber.
  • several reagent delivery ports allow access to the perfusion chamber. For example, one of the ports may be utilized to allow the input of the aqueous sequencing reagents, while another port allows these reagents (and any reaction products) to be withdrawn from the perfusion chamber.
  • the perfusion chamber 200 contains a substrate to which a plurality of anchor primers have been attached.
  • This can be a planar substrate containing one or more anchored primers in anchor pads formed at the termini of a bundled fiber optic arrays. The latter substrate surface is discussed in more detail below.
  • the perfusion chamber allows for a uniform, linear flow of the required sequencing reagents, in aqueous solution form, over the amplified nucleic acids and allows for the rapid and complete exchange of these reagents. Thus, it is suitable for performing pyrophosphate-based sequencing reaction.
  • the perfusion chamber can also be used to prepare the anchor primers and perform amplification reactions, e.g., the RCA reactions described herein.
  • the perfusion chamber is linked to an imaging system 230 , which includes a CCD system in association with conventional optics or a fiber optic bundle.
  • an imaging system 230 which includes a CCD system in association with conventional optics or a fiber optic bundle.
  • a 100 ⁇ m diameter lens for CCD imaging is preferably placed 1 cm above the slide.
  • fiber-optic based imaging it is preferable to incorporate the optical fibers directly into the cover slip.
  • the imaging system 230 is used to collect light from the reactors on the substrate surface. Light can be imaged, for example, onto a CCD using a high sensitivity low noise apparatus known in the art.
  • the imaging system is linked to a computer control and data collection system 240 .
  • a computer control and data collection system is also linked to the conduit 200 to control reagent delivery.
  • FIG. 3 An example of a perfusion chamber of the present invention is illustrated in FIG. 3.
  • the perfusion chamber includes a sealed compartment with transparent upper and lower slide. It is designed to allow linear flow of solution over the surface of the substrate surface and to allow for fast exchange of reagents. Thus, it is suitable for carrying out, for example, the pyrophosphate sequencing reactions. Laminar flow across the perfusion chamber can be optimized by decreasing the width and increasing the length of the chamber.
  • the perfusion chamber is preferably detached form the imaging system while it is being prepared and only placed on the imaging system when sequencing analyses is performed.
  • the solid support i.e., a DNA chip or glass slide
  • a metal or plastic housing which may be assembled and disassembled to allow replacement of said solid support.
  • the lower side of the solid support of the perfusion chamber carries the reaction center array and, with a traditional optical-based focal system, a high numerical aperture objective lens is used to focus the image of the reaction center array onto the CCD imaging system.
  • the photons generated by the pyrophosphate sequencing reaction are captured by the CCD only if they pass through a focusing device (e.g., an optical lens or optical fiber) and are focused upon a CCD element. However, the emitted photons should escape equally in all directions.
  • a planar array e.g., a DNA chip
  • the optical fiber bundle can also be placed against its back surface, eliminating the need to “image” through the depth of the entire reaction/perfusion chamber.
  • the solid support is coupled to a bundle of optical fibers, which are used to detect and transmit sequence reaction of byproducts.
  • the total number of optical fibers within the bundle may be varied so as to match the number of individual arrays utilized in the sequencing reaction.
  • the number of optical fibers incorporated into the bundle is designed to match the CCD (i.e., approximately 30 mm ⁇ 30mm) so as to allow 1:1 imaging.
  • Commercially-available optical fiber bundles range from 25 cm ⁇ 25 cm to 10 ⁇ m in diameter.
  • the desired number of optical fibers are initially fused into a bundle, the terminus of which is cut and polished so as to form a “wafer” of the required thickness (e.g., 1.5 mm).
  • the resulting optical fiber wafers possess similar handling properties to that of a plane of glass.
  • the individual fibers can be any size diameter (e.g., 3 ⁇ m to 100 ⁇ m).
  • the planar support is omitted and the anchor primers are linked directly to the termini of the optical fibers.
  • the anchor primers are attached to termini that are cavitated as shown schematically in FIG. 4.
  • the termini are treated, e.g., with acid, to form a hemispherical shape indentation, or cavitation, that ranges from approximately one-half the depth of an individual optical fiber up to 2- to 3-times the diameter of the fiber.
  • the cavity is preferably 50 ⁇ m deep.
  • Cavities can be introduced into the termini of the fibers by placing one side of the optical fiber wafer into an acid bath for a variable amount of time. The amount of time can vary depending upon the overall depth of the reaction cavity desired (see e.g., Walt, et al., 1996. Anal. Chem. 70: 1888).
  • Several methods are known in the art for attaching molecules (and detecting the attached molecules) in the cavities etched in the ends of fiber optic bundles. See, e.g., Michael, et al., Anal. Chem. 70: 1242-1248 (1998); Ferguson, et al., Nature Biotechnology 14: 1681-1684 (1996); Healey and Walt, Anal. Chem. 69: 2213-2216 (1997).
  • a pattern of reactive sites can also be created in the microwell, using photolithographic techniques similar to those used in the generation of a pattern of reaction pads on a planar support. See, Healey, et al., Science 269: 1078-1080 (1995); Munkholm and Walt, Anal. Chem. 58: 1427-1430 (1986), and Bronk, et al., Anal. Chem. 67: 2750-2757 (1995).
  • the opposing side of the optical fiber wafer i.e., the non-etched side
  • is highly polished so as to allow optical-coupling (e.g., by immersion oil or other optical coupling fluids) to a second, optical fiber bundle.
  • This second optical fiber bundle exactly matches the diameter of the optical wafer containing the reaction chambers, and serve to act as a conduit for the transmission of the photons, generated by the pyrophosphate sequencing reaction, to its attached CCD imaging system or camera.
  • the individual optical fibers utilized to generate the fused optical fiber bundle/wafer are larger in diameter (i.e., 6 ⁇ m to 12 ⁇ m) than those utilized in the optical imaging system (i.e., 3 ⁇ m).
  • the optical imaging fibers can be utilized to image a single reaction site.
  • the etched, hemispherical geometry allows for simultaneously reducing background signal from the PP i released from adjacent anchor pads.
  • delivery of the various sequencing reagents in acid-etched optical fiber bundle embodiment is performed by immersion of the acid-etched cavities, alternately, into dNTP/APS/sulfurylase reagents and then, subsequently, into the apyrase reagents to facilitate the degradation of any remaining dNTPs.
  • Solid-phase pyrophosphate sequencing was initially developed by combining a solid-phase technology and a sequencing-by-synthesis technique utilizing bioluminescence (see e.g., Ronaghi, et al., 1996. Real-time DNA sequencing using detection of pyrophosphate release. Anal. Biochem. 242: 84-89).
  • an immobilized, primed DNA strand is incubated with DNA polymerase, ATP sulfurylase, and luciferase.
  • stepwise nucleotide addition with intermediate washing the event of sequential polymerization can be followed.
  • a remarkable increase in signal-to-noise ratio was obtained by the use of ⁇ -thio dATP in the system.
  • This DATP analog is demonstrated to be efficiently incorporated by DNA polymerase while being silent for luciferase, allowing the sequencing reaction to be performed in real-time.
  • sequencing of a PCR product using streptavidin-coated magnetic beads as a solid support was presented. However, it was found that the loss of the beads during washing, which was performed between each nucleotide and enzyme addition, was the limiting factor to sequence longer stretches.
  • Pyrophosphate (PPi)-producing reactions can be monitored by a very sensitive technique based on bioluminescence (see e.g., Nyrén, et al., 1996. pp. 466-496 ( Proc. 9 th Inter. Symp. Biolumin. Chemilumin .).
  • bioluminometric assays rely upon the detection of the PPi released in the different nucleic acid-modifying reactions. In these assays, the PPi which is generated is subsequently converted to ATP by ATP sulfurylase and the ATP production is continuously monitored by luciferase.
  • the PPi is generated when a nucleotide is incorporated into a growing nucleic acid chain being synthesized by the polymerase.
  • a DNA polymerase is utilized to generate PPi during a pyrophosphate sequencing reaction (see e.g., Ronaghi, et al, 1998. Doctoral Dissertation , The Royal Institute of Technology, Dept. of Biochemistry (Stockholm, Sweden)), it is also possible to use reverse transcriptase (see e.g., Karamohamamed, et al., 1996. pp. 319-329 ( Proc. 9 th Inter. Symp. Biolumin. Chemilumin .) or RNA polymerase (see e.g., Karamohamamed, et al., 1998. BioTechniques 24: 302-306) to follow the polymerization event.
  • a bioluminometric primer extension assay has been utilized to examine single nucleotide mismatches at the 3′-terminus (see e.g., Nyrén, et al., 1997. Anal. Biochem. 244: 367-373).
  • a phage promoter is typically attached onto at least one of the arbitrary primers and, following amplification, a transcriptional unit may be obtained which can then be subjected to stepwise extension by RNA polymerase.
  • the transcription-mediated PPi-release can then be detected by a bioluminometric assays (e.g., ATP sulfurylase-luciferase).
  • the rate of polymerization could be decreased by 5-fold to 13-fold, thus causing a delay in the incorporation of correct nucleotides by the DNA polymerase after the primer comprising a mismatch at the 3′-termini.
  • the rate of DNA synthesis is comparable with the rate observed for. a normal template/primer.
  • Single-base detection by this technique has been improved by incorporation of apyrase to the system, which functions to degrade the nucleotide to a concentration far below the K m of the DNA polymerase.
  • apyrase minimizes further extension upon contact with a mismatched base, and thereby simplifies the data analysis.
  • the above-described technique provides a rapid and real-time analysis for applications in the areas of mutation detection and single-nucleotide polymorphism (SNP) analysis.
  • the pyrophosphate sequencing system takes advantage of the cooperativity of several enzymes to monitor DNA synthesis. Parameters such as stability, fidelity, specificity, sensitivity, K M and K CAT are of paramount importance for the optimal performance of the enzymes used in the system.
  • the activity of the detection enzymes i.e., sulfurylase and luciferase
  • the activity of the detection enzymes generally remain constant during the sequencing reaction, and are only very slightly inhibited by high amounts of products (see e.g., Ronaghi, et al., 1998. Doctoral Dissertation, The Royal Institute of Technology, Dept. of Biochemistry (Stockholm, Sweden)).
  • Sulfurylase converts PPi to ATP in approximately 2.0 seconds, and the generation of light by luciferase takes place in less than 0.2 seconds. The most critical reactions are the DNA polymerization and the degradation of nucleotides.
  • the value of the enzymes utilized in the pyrophosphate sequencing methodology are listed below: Enzyme K M ( ⁇ M) K CAT (S ⁇ 1 ) Klenow 0.18 (dTTP) 0.92 T 7 DNA Polymerase 0.36 (dTTP) 0.52 ATP Sulfurylase 0.56 (APS); 7.0 (PPi) 38 Firefly Luciferase 20 (ATP) 0.015 Apyrase 120 (ATP); 260 (ADP) 500 (ATP)
  • an induced-fit binding mechanism in the polymerization step provides a very efficient selectivity for the correct dNTP with a net contribution, approaching a fidelity of 1 ⁇ 10 5 to 1 ⁇ 10 6 (see e.g., Wong, et al., 1991. An induced-fit kinetic mechanism for DNA replication fidelity. Biochemistry 30: 526-537).
  • exonuclease-deficient (exo-) polymerases such as exo-Klenow or Sequenase®, catalyze the incorporation of a nucleotide only in the presence of a complementary dNTP, confirming a high fidelity of these enzymes even in the absence of proof-reading exonuclease activity.
  • exo- polymerases the K M and K CAT for a the incorporation of a single nucleotide is lower than that of the incorporation of several (see e.g., Van Draanen, et al., 1992. Beta-L-thymidine 5′-triphosphate analogs as DNA polymerase substrates. J. Biol.
  • nucleotide-degrading enzyme must possess the following properties: firstly, the enzyme must hydrolyze all deoxynucleotide triphosphates at approximately the same rate. Secondly, it should also hydrolyze ATP to prevent the accumulation of ATP between cycles.
  • the time for nucleotide degradation by the nucleotide-degrading enzyme must be lower than nucleotide incorporation by the polymerase. It is also important that the yield of primer-directed incorporation is as close to 100% as possible before the nucleotide-degrading enzyme has degraded the nucleotide to a concentration below the K M of the polymerase. Changes in other parameters, such as pH, temperature, and ionic concentration may also alter the kinetics of the different enzymes in the system. However, the enzymes typically utilized in the pyrophosphate sequencing system show high stability within a rather broad range of these parameters for several hours (see e.g., Ronaghi, et al., 1998. Doctoral Dissertation, The Royal Institute of Technology, Dept. of Biochemistry (Stockholm, Sweden)).
  • N(0) is the DNA with no nucleotides added
  • N(1) has 1 nucleotide added
  • N(2) has 2 nucleotides added
  • the pseudo-first-order rate constants which relate the concentrations of molecular species are: N(n) ⁇ N(n + 1) + PP 1 k N PP 1 ⁇ ATP k P ATP ⁇ L k A
  • the diffusion constants D p for PP i and D A for ATP must also be specified. These values may be estimated from the following exemplar diffusion constants for biomolecules in a dilute water solution (see Weisiger, 1997. Impact of Extracellular and Intracellular Diffusion on Hepatic Uptake Kinetics Department of Medicine and the Liver Center, University of California, San Francisco, Calif., USA, dickw@itsa.ucsf.edu, http://dickw.ucsf.edu/Dapers/goresky97/chapter.html). Molecule D/10 ⁇ 5 cm 2 /sec Method Original Reference Albumin 0.066 lag time 1 Albumin 0.088 light scattering 2 Water 1.940 NMR 3
  • Original Reference 1 is: Longsworth, 1954. Temperature dependence of diffusion in aqueous solutions, J. Phys. Chem. 58: 770-773;
  • Original Reference 2 is: Gaigalas, et al., 1992. Diffusion of bovine serum albumin in aqueous solutions, J. Phys. Chem. 96: 2355-2359;
  • Original Reference 3 is: Cheng, 1993. Quantitation of non-Einstein diffusion behavior of water in biological tissues by proton NMR diffusion imaging: Synthetic image calculations, Magnet. Reson. Imaging 11: 569-583.
  • the molecular weight of PP i is 174 amu. Based upon the aforementioned exemplar values, a diffusion constant of approximately 0.7 ⁇ 10 ⁇ 5 cm 2 /sec for PP i is expected.
  • V max k turnover [E T ]
  • [S] is the concentration of substrate
  • [E] is the concentration of free enzyme
  • [ES] is the concentration of the enzyme-substrate complex
  • reaction times are at least as fast as the solution-phase pyrophosphate-based sequencing described in the literature. That rate that a substrate is converted into product is
  • the effective concentration of substrate may be estimated from the size of a replicated DNA molecule, at most (10 ⁇ m) 3 and the number of copies (approximately 10,000), yielding a concentration of approximately 17 nm. This is this is smaller than the K M for the enzymes described previously, and therefore the rate can be estimated to be
  • the first step in the pyrophosphate sequencing reaction i.e., incorporation of a new nucleotide and release of PP i
  • the preferred reaction conditions are: 1 pmol DNA, 3 pmol polymerase, 40 pmol dNTP in 0.2 ml buffer.
  • the K M for nucleotide incorporation for the Klenow fragment of DNA polymerase I is 0.2 ⁇ M and for Sequenase 2.0TM (Promega Biotech, Madison, Wis.) is 0.4 ⁇ M, and complete incorporation of 1 base is less than 0.2 sec (see e.g., Ronaghi, et al., 1996. Real-Time DNA Sequencing Using Detection of Pyrophosphate Release, Anal. Biochem. 242: 84-89) with a polymerase concentration of 1.5 nM.
  • the time required for the nucleotide addition reaction is no greater than 0.2 sec per nucleotide.
  • the reaction is allowed to proceed for a total of T seconds, then nucleotide addition should be sufficiently rapid that stretches of up to (T/0.2) identical nucleotides should be completely filled-in by the action of the polymerase.
  • the rate-limiting step of the pyrophosphate sequencing reaction is the sulfurylase reaction, which requires a total of approximately 2 sec to complete. Accordingly, a total reaction time which allows completion of the sulfurylase reaction, should be sufficient to allow the polymerase to “fill-in” stretches of up to 10 identical nucleotides.
  • regions of 10 or more identical nucleotides have been demonstrated to occur with a per-nucleotide probability of approximately 4 ⁇ 10 , which is approximately 1 ⁇ 10 ⁇ 6 .
  • each of which will be extended at least 30 nt. and preferably 100 nt., it is expected that approximately one run of 10 identical nucleotides will be present.
  • runs of identical nucleotides should not pose a difficulty in the practice of the present invention.
  • the overall size of the resulting DNA molecule is, preferably, smaller than the size of the anchoring pads (i.e., 10 ⁇ m) and must be smaller than the distance between the individual anchoring pads (i. e., 100 ⁇ m).
  • [PP i ] is approximately 0.17 fmol in 5 ⁇ l, or 0.03 nM.
  • the fraction of PP i which is bound to the enzyme is [E]/K M , where [E] is the concentration of free enzyme. Since the enzyme concentration is much larger than the PP i concentration, the total enzyme concentration alone, may be used in the calculations.
  • the root mean square (RMS) distance it diffuses in each direction is approximately 2D p /k p , or 2.8 ⁇ 10 3 ⁇ m 2 .
  • the RMS distance in each direction is 53 ⁇ m. This value indicates that each of the individual anchor primers must be more than 50 ⁇ m apart, or PP i which is released from one anchor could diffuse to the next, and be detected.
  • Another method which may be used to explain the aforementioned phenomenon is to estimate the amount of PP i over a first anchor pad that was generated at said first anchor pad relative to the amount of PP i that was generated at a second anchor pad and subsequently diffused over to the location of said first anchor pad.
  • a is approximately equal to 10 ⁇ m and b is approximately equal to 100 ⁇ m.
  • the amount of PP i which is present over said first anchor pad may be described by: exp( ⁇ k p t)[1 ⁇ exp( ⁇ a 2 /2D p t)] and the amount of PP i present over the second anchor pads may be mathematically-approximated by: (1 ⁇ 3)exp( ⁇ k p t)[pa 2 /b 2 ]exp( ⁇ b 2 /2D p t).
  • the prefactor 1 ⁇ 3 assumes that 1 ⁇ 4 of the DNA sequences will incorporate 1 nucleotide, 1 ⁇ 4 of these will then incorporate a second nucleotide, etc., and thus the sum of the series is 1 ⁇ 3.
  • the anchor pads must be placed no closer than approximately 50 ⁇ m apart, and preferable are at least 3-times further apart (i.e., 150 ⁇ m).
  • a second approach to decrease the distance between anchor pads is to increase the concentration of sulfurylase in the reaction mixture.
  • the reaction rate k p is directly proportional to the sulfurylase concentration, and the diffusion distance scales as k p ⁇ 1 ⁇ 2 . Therefore, if the sulfurylase enzyme concentration is increased by a factor of 4-times, the distance between individual anchor pads may be concomitantly reduced by a factor of 2-times.
  • a third approach is to increase the effective concentration of sulfurylase (which will also work for other enzymes described herein) by binding the enzyme to the surface of the anchor pads.
  • the anchor pad can be approximated as one wall of a cubic surface enclosing a sequencing reaction center. Assuming a 10 ⁇ m ⁇ 10 ⁇ m surface for the pad, the number of molecules bound to the pad to produce a concentration of a 1 ⁇ M is approximately 600,000 molecules.
  • the sulfurylase concentration in the assay is estimated as 5 nM.
  • the number bound molecules to reach this effective concentration is about 3000 molecules. Thus, by binding more enzyme molecules, a greater effective concentration will be attained. For example, 10,000 molecules could be bound per anchor pad.
  • each sulfurylase molecule occupies a total area of 65 nm 2 on a surface. Accordingly, anchoring a total of 10,000 sulfurylase enzyme molecules on a surface (i.e., so as to equal the 10,000 PP i released) would require 1.7 ⁇ m 2 . This value is only approximately 2% of the available surface area on a 10 ⁇ m ⁇ 10 ⁇ m anchor pad. Hence, the concentration of the enzyme may be readily increased to a much higher value.
  • a fourth approach to allow a decrease in the distance between individual anchor pads is to utilize one or more agents to increase the viscosity of the aqueous-based, pyrophosphate sequencing reagents (e.g., glycerol, polyethylene glycol (PEG), and the like) so as to markedly increase the time it takes for the PPi to diffuse.
  • agents e.g., glycerol, polyethylene glycol (PEG), and the like
  • these agents will also concomitantly increase the diffusion time for other non-immobilized components within the sequencing reaction, thus slowing the overall reaction kinetics.
  • the use of these agents may also function to chemically-interfere with the sequencing reaction itself.
  • a fifth, and preferred, methodology to allow a decrease in the distance between individual anchor pads is to conduct the pyrophosphate sequencing reaction in a spatial-geometry which physically-prevents the released PP i from diffusing laterally.
  • uniform cavities which are generated by acid-etching the termini of optical fiber bundles, may be utilized to prevent such lateral diffusion of PPi (see Michael, et al., 1998. Randomly Ordered Addressable High-Density Optical Sensor Arrays, Anal. Chem. 70: 1242-1248).
  • the important variable involves the total diffusion time for the PP i to exit a cavity of height h, wherein h is the depth of the etched cavity.
  • delivery of the various sequencing reagents in acid-etched optical fiber bundle embodiment is performed by immersion of the acid-etched cavities, alternately, into dNTP/APS/sulfurylase reagents and then, subsequently, into the apyrase reagents to facilitate the degradation of any remaining dNTPs.
  • reaction time 1/k A
  • the reaction time has been shown to be 0.2 seconds. Because this reaction time is much lower than the time which the PP i is free to diffuse, it does not significantly alter any of the aforementioned conclusions regarding the assay geometry and conditions utilized in the present invention.
  • luciferase In order to mitigate the generation of background light, it is preferable to “localize” (i.e., anchoring or binding) the luciferase in the region of the DNA sequencing templates. It is most preferable to localize the luciferase to a region that is delineated by the distance a PP i molecule can diffuse before it forms ATP.
  • Methods for binding luciferase to a solid support matrix are well-known in the literature (see e.g., Wang, et al., 1997. Specific Immobilization of Firefly Luciferase through a Biotin Carboxyl Carrier Protein Domain, Analytical Biochem. 246: 133-139).
  • the luciferase is anchored within a 50 ⁇ m distance of the DNA strand. It should be noted, however, that it would be preferable to decrease the diffusion time and thus to further limit the surface area which is required for luciferase binding.
  • the surface area of luciferase has been computed at 50 nm 2 .
  • 1000 molecules would occupy a total area of 0.05 ⁇ m 2 . From these calculations it becomes readily apparent that a plethora of luciferase molecules may be bound to the anchor pad, as the area of each anchor pad area is 100 ⁇ m 2 .
  • each nucleotide takes approximately 3 seconds in toto, to sequence (i.e., 0.5 seconds to add a nucleotide; 2 seconds to make ATP; 0.2 seconds to get fluorescence). Accordingly, a cycle time of approximately 60 seconds per nucleotide is reasonable, requiring approximately 30 minutes per experiment to generate 30 nucleotides of information per sequencing template.
  • a polymerase may be developed (e.g., through the use of protein fusion and the like) which possesses the ability to generate light when it incorporates a nucleotide into a growing DNA chain.
  • a sensor may be developed which directly measures the production of PP i in the sequencing reaction. As the production of PP i changes the electric potential of the surrounding buffer, this change could be measured and calibrated to quantitate the concentration of PP i produced.
  • the polymerase-mediated incorporation of dNTPs into the nucleotide sequence in the pyrophosphate sequencing reaction causes the release of an inorganic pyrophosphate (PPi) moiety which, in turn, through catalysis by luciferase, causes the release of a photon (i.e., light).
  • PPi inorganic pyrophosphate
  • the photons generated by the pyrophosphate sequencing reaction may subsequently be “captured” and quantitated by a variety of methodologies including, but not limited to: a photomultiplier tube, charge-coupled display (CCD), absorbance photometer, a luminometer, and the like.
  • the photons generated by the pyrophosphate sequencing reaction are captured by the CCD only if they pass through a focusing device (e.g., an optical lens or optical fiber) and are focused upon a CCD element.
  • a focusing device e.g., an optical lens or optical fiber
  • the emitted photons should escape equally in all directions.
  • the photons are dispersed over an area of which is equal to 4 ⁇ r 2 .
  • the fraction of photons which pass through the lens is described by: (1 ⁇ 2)[1 ⁇ (1+b 2 /4r 2 ) ⁇ 1 ⁇ 2 ].
  • the fraction which pass through the lens may then be described by: b 2 /16r 2 .
  • this fraction of photons is 6 ⁇ 10 ⁇ 6 .
  • PP i molecules For each nucleotide addition, it is expected that approximately 10,000 PP i molecules will be generated and, if all are converted by sulfurylase and luciferase, these PPi will result in the emission of approximately 1 ⁇ 10 4 photons.
  • a planar array e.g., a DNA chip
  • the fraction collected is found to be 0.15, which equates to the capture of approximately 1 ⁇ 10 3 photons. This value would be sufficient to provide an adequate signal.
  • the sequence acquisition software acquires and analyzes the data during the pyrophosphate sequencing cycle. Prior to beginning a given sequencing experiment, a bin of pixels containing each individual reaction center is determined. During each sequencing cycle, four “images” of the entire array are produced, and each image corresponds to excitation of one of the four, fluorescently-labeled nucleotide bases A, C, G, or T (or U). For each reaction center bin, all of the four images are analyzed to determine which nucleotide species has been incorporated at that reaction center during that cycle. As described above, the reaction center bin corresponding to a certain reaction center contains a 10 ⁇ 10 array of pixels.
  • the total number of photons produced by the single fluorophore in that reaction center is determined by the summation of each pixel value in the array.
  • the sums of the reaction center bins from each of the four images are compared, and the image that produces a significant sum corresponds to the newly incorporated base at that reaction center.
  • the images are processed for each of the reaction centers and an array of incorporated nucleotides is recorded. Such processing is capable of being rapidly performed in real-time with modem image processing computers.
  • Multiple “reads” of individual reaction center arrays may be necessary during the detection step to ensure that the four nucleotides are properly distinguished. Exposure times can be as rapid as 100 msec, with the readout time of the CCD chip being on the order of 250 msec. Thus, the maximum time needed for four complete reads of the array is 1.5 seconds. The total time for a given cycle, including reagent addition, removal, and washes, is certainly less than 10 seconds. Accordingly, a sequencing apparatus consisting of an array of 10,000 reaction centers (i.e., a 100 ⁇ 100 array) is able to detect at least 360 bases per site per hour, or 3.6 Megabases per hour of total sequence, as a conservative estimate. This rate is significantly faster than those of traditional sequencing methodologies.
  • the methods of the present invention do not require the time-consuming processes of initial sample amplification (e.g., cloning or PCR), and gel electrophoresis.
  • initial sample amplification e.g., cloning or PCR
  • gel electrophoresis e.g., gel electrophoresis
  • a nucleic acid sample is sheared prior to inclusion in a reaction center. Once these fragments have been sequenced, sequence analysis software is used to assemble their sequences into contiguous stretches. Many algorithms exist in the art that can compare sequences and deduce their correct overlap. New algorithms have recently been designed to process large amounts of sequence data from shotgun (random) sequencing approaches.
  • an algorithm initially reduces the amount of data to be processed by using only two smaller sequences derived from either end of the sequence deduced from a single reaction center in a given experiment.
  • This approach has been proposed for use in shotgun sequencing of the human genome (see e.g., Rawlinson, et al., 1996 J. Virol 70: 8833-8849; Venter, et al., 1998. Science 280: 1540-1542). It employs algorithms developed at the Institute for Genome Research (TIGR; see e.g., Sutton, et al., 1995. Genome Sci. Technol. 1: 9-16).
  • raw data is compressed into a fingerprint of smaller words (e.g., hexanucleotide restriction enzyme sites) and these fingerprints can be compared and assembled into larger continuous blocks of sequence (i.e., contigs).
  • This technique is similar to that used to deduce overlapping sequences after oligonucleotide hybridization (see e.g., Idury and Waterman, 1995. J. Comput. Biol. 2: 291-306).
  • Yet another embodiment uses existing sequence data, from genetic or physical linkage maps, to assist the assembly of new sequence data from whole genomes or large genomic pieces.
  • termini of a thin wafer fiber optic array are cavitated by inserting the termini into acid as described by Healey et al., Anal. Chem. 69: 2213-2216 (1997).
  • a thin layer of a photoactivatable biotin analog is dried onto the cavitated surface as described Hengsakul and Cass ( Biocongiugate Chem. 7: 249-254, 1996) and exposed to white light through a mask to create defined pads, or areas of active biotin.
  • avidin is added and allowed to bind to the biotin.
  • Biotinylated oligonucleotides are then added.
  • the avidin has free biotin binding sites that can anchor biotinylated oligonucleotides through a biotin-avidin-biotin link.
  • the pads are approximately 10 ⁇ m on a side with a 100 ⁇ m spacing. Oligonucleotides are added so that approximately 37% of the pads include one anchored primer. On a 1 cm surface are deposited 10,000 pads, yielding approximately 3700 pads with a single anchor primer. Sulfurylase, apyrase, and luciferase are also attached to the cavitated substrate using biotin-avidin.
  • a library of open circle library templates is prepared from a population of nucleic acids suspected of containing a single nucleotide polymorphism on a 70 bp Sau3A1-MspI fragment.
  • the templates include adapters that are complementary to the anchor primer, a region complementary to a sequencing primer, and an insert sequence that is to be characterized.
  • the library is generated using Sau3A1 and MspI to digest the genomic DNA. Inserts approximately 65-75nucleotides are selected and ligated to adaptor oligonucleotides 12 nucleotides in length.
  • the adapter oligonucleotides have have sequences complementary to sequences to an anchor primers linked to a substrate surface as described in Example 1.
  • the library is annealed to the array of anchor primers.
  • a DNA polymerase is added, along with dNTPS, and rolling circle replication is used to extend the anchor primer.
  • the result is a single DNA strand, still anchored to the solid support, that is a concatenation of multiple copies of the circular template. 10,000 or more copies of circular templates in the hundred nucleotide size range.
  • the fiber optic array wafer containing amplified nucleic acids as described in Example 2 is placed in a perfusion chamber and attached to a bundle of fiber optic arrays, which are themselves linked to a 16 million pixel CCD cameras.
  • a sequencing primer is delivered into the perfusion chamber and allowed to anneal to the amplified sequences.
  • the sequencing primer primes DNA synthesis extending into the insert suspected of having a polymorphism, as shown in FIG. 1.
  • the sequencing primer is first extended by delivering into the perfusion chamber, in succession, a wash solution, a DNA polymerase, and one of dTTP, dGTP, dCTP, or APS (a dATP analog).
  • the sulfurylase, luciferase, and apyrase, attached to the termini convert any PPi liberated as part of the sequencing reaction to detectable light.
  • Light is allowed to collect for 3 seconds by a CCD camera linked to the fiber imaging bundle, after which additional wash solution is added to the perfusion chamber for 10 seconds.
  • the next nucleotide is then added, along with polymerase, thereby repeating the cycle.
  • the collected light image is transferred from the CCD camera to a computer.
  • Light emission is analyzed by the computer and used to determine whether the corresponding dNTP has been incorporated into the extended sequence primer.
  • Addition of dNTPS and pyrophosphate sequencing reagents is repeated until the sequence of the insert region containing the suspected polymorphism is obtained.
  • the sequence of the amplified nucleic acid can be determined using by products of RNA synthesis.
  • an RNA transcript is generated from a promoter sequence present in the circular nucleic acid template library.
  • Suitable promoter sites and their cognate RNA polymerases include RNA polymerases from E. coli, the RNA polymerase from the bacteriophage T 3 , the RNA polymerase from the bacteriophage T 7 , the RNA polymerase from the bacteriophage SP6, and the RNA polymerases from the viral families of bromoviruses, tobamoviruses, tombusvirus, lentiviruses, hepatitis C-like viruses, and picornaviruses.
  • RNA transcript a predetermined NTP, i.e., an ATP, CTP, GTP, or UTP, is incubated with the template in the presence of the RNA polymerase. Incorporation of the test NTP into a nascent RNA strand can be determined by assaying for the presence of PPi using the enzymatic detection discussed herein.
  • NTP i.e., an ATP, CTP, GTP, or UTP

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Studio Devices (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Optical Measuring Cells (AREA)
  • Investigating Or Analysing Materials By Optical Means (AREA)

Abstract

Disclosed herein are methods and apparatuses for sequencing a nucleic acid. The method includes annealing a population of circular nucleic acid molecules to a plurality of anchor primers linked to a solid support, and amplifying those members of the population of circular nucleic acid molecules which anneal to the target nucleic acid, and then sequencing the amplified molecules by detecting the presence of a sequence byproduct.

Description

    FIELD OF THE INVENTION
  • The present invention relates to methods and apparatuses for determining the sequence of a nucleic acid. [0001]
    • BACKGROUND OF THE INVENTION
  • Many diseases are associated with particular DNA sequences. The DNA sequences are often referred to as DNA sequence polymorphisms to indicate that the DNA sequence associated with a diseased state differs from the corresponding DNA sequence in non-afflicted individuals. DNA sequence polymorphisms can include, e.g., insertions, deletions, or substitutions of nucleotides in one sequence relative to a second sequence. An example of a particular DNA sequence polymorphism is 5′-ATCG-3′, relative to the [0002] sequence 5′-ATGG-3′. The first nucleotide ‘G’ in the latter sequence has been replaced by the nucleotide ‘C’ in the former sequence. The former sequence is associated with a particular disease state, whereas the latter sequence is found in individuals not suffering from the disease. Thus, the presence of the nucleotide sequence ‘5-ATCG-3’ indicates the individual has the particular disease. This particular type of sequence polymorphism is known as a single-nucleotide polymorphism, or SNP, because the sequence difference is due to a change in one nucleotide.
  • Techniques which enable the rapid detection of as little as a single DNA base change is therefore important methodologies for use in genetic analysis. Because the size of the human genome is large, on the order of 3 billion base pairs, techniques for identifying polymorphisms must be sensitive enough to specifically identify the sequence containing the polymorphism in a potentially large population of nucleic acids. [0003]
  • Typically a DNA sequence polymorphism analysis is performed by isolating DNA from an individual, manipulating the isolated DNA, e.g., by digesting the DNA with restriction enzymes and/or amplifying a subset of sequences in the isolated DNA. The manipulated DNA is then examined further to determine if a particular sequence is present. [0004]
  • Commonly used procedures for analyzing the DNA include electrophoresis. A common application of electrophoresis includes agarose or polyacrylamide gel electrophoresis. DNA sequences are inserted, or loaded, on the gels and subjected to an electric field. Because DNA carries a uniform negative charge, DNA will migrate through the gel based on a charge/mass ratio upon application of the electrical field. Smaller DNA molecules will migrate more rapidly through the gel than larger fragments. After electrophoresis has been continued for a sufficient length of time, the DNA molecules in the initial population of DNA sequences will have separated according to their relative sizes. [0005]
  • Particular DNA molecules can then be detected using a variety of detection methodologies. For some applications, particular DNA sequences are identified by the presence of detectable tags, such as radioactive labels, attached to specific DNA molecules. [0006]
  • Electrophoretic-based separation analyses can be less desirable for applications in which it is desirable to rapidly, economically, and accurately analyze a large number of nucleic acid samples for particular sequence polymorphisms. For example, electrophoretic-based analysis can require a large amount of input DNA. In addition, processing the large number of samples required for electrophoretic-based nucleic acid based analyses can be labor intensive. [0007]
  • Recently, automated electrophoresis systems have become available. However, electrophoresis can be ill-suited for applications such as clinical sequencing, where relatively cost-effective units with high throughput are needed. Thus, the need for non-electrophoretic methods for sequencing is great. For many applications, electrophoreses is used in conjunction with DNA sequence analysis. [0008]
  • Several alternatives to electrophoretic-based sequencing have been described. These include scanning tunnel electron microscopy, sequencing by hybridization, and single molecule detection methods. [0009]
  • Another alternative to electrophoretic-based separation is analysis is solid substrate-based nucleic acid analyses. These methods typically rely upon the use of large numbers of nucleic acid probes affixed to different locations on a solid support. These solid supports can include, e.g., glass surfaces, plastic microtiter plates, plastic sheets, thin polymer, semi-conductors. The probes can be, e.g., adsorbed or covalently attached to the support, or can be microencapsulated or otherwise entrapped within a substrate membrane or film. [0010]
  • Substrate-based nucleic acid analyses can include applying a sample nucleic acid known or suspected of containing a particular sequence polymorphism to an array of probes attached to the solid substrate. The nucleic acids in the population are allowed to hybridize to complementarty sequences attached to the substrate, if present. Hybridizing nucleic acid sequences are then detected in a detection step. [0011]
  • Solid support matrix-based hybridization and sequencing methodologies can require a high sample-DNA concentration and can be hampered by the relatively slow hybridization kinetics of nucleic acid samples with immobilized oligonucleotide probes. Often, only a small amount of template DNA is available, and it can be desirable to have high concentrations of the target nucleic acid sequence. Thus, substrate based detection analyses often include a step in which copies of the target nucleic acid, or a subset of sequences in the target nucleic acid, is amplified. Methods based on the Polymerase Chain Reaction (PCR), e.g., can increase a small number of probes targets by several orders of magnitude in solution. However, PCR can be difficult to incorporate into a solid-phase approach because the amplified DNA is not immobilized onto the surface of the solid support matrix. [0012]
  • Solid-phase based detection of sequence polymorphisms has been described. An example is a “mini-sequencing” protocol based upon a solid phase principle described by Hultman, et al., 1988. [0013] Nucl. Acid. Res. 17: 4937-4946; Syvanen, et al., 1990. Genomics 8: 684-692). In this study, the incorporation of a radiolabeled nucleotide was measured and used for analysis of a three-allelic polymorphism of the human apolipoprotein E gene. However, such radioactive methods are not well-suited for routine clinical applications, and hence the development of a simple, highly sensitive non-radioactive method for rapid DNA sequence analysis has also been of great interest.
    • SUMMARY OF THE INVENTION
  • The invention is based in part on the discovery of a highly sensitive method for determining the sequences of nucleic acids attached to solid substrates, and of novel substrate services for analyzing nucleic acid sequences. [0014]
  • Accordingly, in one aspect, the invention includes a substrate for analyzing a nucleic acid. The substrate includes a fiber optic surface onto which has been affixed one or more nucleic acid sequences. The fiber optic surface can be cavitated, e.g., a hemispherical etching of the opening of a fiber optic. The substrate can in addition include a plurality of bundled fiber optic surfaces, where one or more of the surfaces have anchored primers. [0015]
  • In another aspect, the invention includes an apparatus for analyzing a nucleic acid sequence. The apparatus can include a perfusion chamber, wherein the chamber includes a nucleic acid substrate, a conduit in communication with the perfusion chamber, an imaging system, e.g., a fiber optic system, in communication with the perfusion chamber; and a data collection system in communication with the imaging system. The substrate can be a planar substrate. In other embodiments, the substrate can be the afore-mentioned fiber optic surface having nucleic acid sequences affixed to its termini. [0016]
  • In a further aspect, the invention includes a method for sequencing a nucleic acid. The method includes providing one or more or more nucleic acid anchor primers linked to a solid support and a plurality of circular nucleic acid templates. The nucleic acid anchor primer is then annealed to at least one of the single-stranded circular templates to yield a primed anchor primer-circular template complex. The primed anchor primer-circular template complex is then combined with a polymerase to generate multiple copies of the circular nucleic acid template. Next, a sequencing primer is annealed to the circular nucleic acid template to yield a primed sequencing primer-circular nucleic acid template complex. The sequence primer is the extended with a polymerase and a predetermined nucleotide triphosphate to yield a sequencing product and a sequencing reaction byproduct, e.g., inorganic pyrophosphate. If the predetermined nucleotide is incorporated into the primer, the sequencing reaction byproduct is generated and then identified, thereby determining the sequence of the nucleic acid. If desired, a additional predetermined nucleotide triphosphates can be added, e.g., sequentially, and the presence or absence of sequence byproducts associated with each reaction can be determined. [0017]
  • In a still further aspect, the invention includes a method for sequencing a nucleic acid by providing one or more nucleic acid anchor primers linked to a plurality of anchor primers linked to a fiber optic surface substrate, e.g., the solid substrate discussed above. [0018]
  • In various embodiments of the apparatuses and methods described herein, the solid substrate includes two or more anchoring primers separated by approximately 10 μm to approximately 200 μm, 50 μm to approximately 150 μm, 100 μm to approximately 150 μm, or 150 μm. The solid support matrix can include a plurality of pads that are covalently linked to the solid support. The surface area of the pads can be, e.g., 10 μm[0019] 2 and one or more pads can be separated from one another by a distance ranging from approximately 50 μm to approximately 150 μm.
  • In preferred embodiments, at least a portion of the circular nucleic acid template is single-stranded DNA. The circular template can be, e.g., an open-circle nucleic acid or a closed circle nucleic acid. The circular nucleic acid template can be, e.g., genomic DNA or RNA, or a cDNA copy thereof. The circular nucleic acid can be, e.g., 10-10,000 or 10-1000, 10-200, 10-100, 10-50, or 20-40 nucleotides in length. [0020]
  • In some embodiments, multiple copies of one or more circular nucleic acids in the population are generated by a polymerase chain reaction. In other embodiments, the primed circular template is extended by rolling circle amplification (RCA) to yield a single-stranded concatamer of the annealed circular nucleic acid template. If desired, the template amplified by rolling circle amplificaion and be further amplified by annealing a reverse primer to the single-stranded concatamer to yield a primed concatamer template and combining the primed concatamer template with a polymerase enzyme to generate multiple copies of the concatamer template. In still further embodiments, the template can be extended by a combination of PCR and RCA-amplification. [0021]
  • In preferred embodiments, sequencing byproduct analyzed is pyrophosphate. When pyrophosphate is used as the detected byproduct, a preferred nucleotide triphosphate for use by the polymerase in extending the primed sequencing primer is a dATP analog, e,g., [0022] adenosine 5′-phosphosulfate (APS).
  • Preferably, the pyrophosphate is detected by contacting the sequencing byproduct with ATP sulfurylase under conditions sufficient to form ATP. The ATP can then be detected, e.g., with an enzyme which generates a detectable product upon reaction with ATP. A preferred enzyme for detecting the ATP is luciferase. If desired, a wash buffer, can be used between addition of various reactants herein. Preferably, apyrase is used to remove, e.g., unreacted dNTP used to extend the sequencing primer. The wash buffer can optionally include apyrase. [0023]
  • The reactants and enzymes used herein, e.g., the ATP sulfurylase, luciferase, and apyrase, can be attached to the solid surface. [0024]
  • The anchor primer sequence can include, e.g. a biotin group, which can link the anchor primer to the solid support via an avidin group attached to the solid support. In some embodiments, the anchor primer is conjugated to a biotin-bovine serum albumin (BSA) moiety. The biotin-BSA moiety can be linked to an avidin-biotin group on the solid support. If desired, the biotin-BSA moiety on the anchor primer can be linked to a BSA group on the solid support in the presence of silane. [0025]
  • In some embodiments, the solid support includes at least one optical fiber. [0026]
  • The disclosures of one or more embodiments of the invention are set forth in the accompanying description below. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. Other features, objects, and advantages of the invention will be apparent from the description and from the claims. In the specification and the appended claims, the singular forms include plural referents unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless expressly stated otherwise, the techniques employed or contemplated herein are standard methodologies well known to one of ordinary skill in the art. The examples of embodiments are for illustration purposes only. All patents and publications cited in this specification are incorporated by reference.[0027]
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 is a schematic illustration of rolling circle based amplification using an anchor primer. [0028]
  • FIG. 2 is a drawing of a sequencing apparatus according to the present invention. [0029]
  • FIG. 3 is a drawing of a perfusion chamber according to the present invention. [0030]
  • FIG. 4 is a drawing of a cavitated fiber optic terminus of the present invention.[0031]
    • DETAILED DESCRIPTION OF THE INVENTION
  • The methods described herein include a sample preparation process in which multiple copies of individual single-stranded nucleic acid molecules, termed anchor primers, are linked to a solid substrate. As is explained in more detail below, a region of the substrate containing at least one linked anchor primer is a anchor pad. A plurality of anchor primers linked on a single solid surface can form an array. [0032]
  • A plurality of nucleic acid template sequences is then annealed to the array to form one or more primed circular templates. The primed circular templates are next amplified. After amplification, a sequencing primer is annealed to the amplified nucleic acid and used to generate a sequencing product. The nucleotide sequence of the sequence product is then determined, thereby allowing for the determination of the nucleic acid. [0033]
  • The methods and apparatuses described herein allow for the determination of nucleic acid sequence information without the need for first cloning a nucleic acid. In addition, the method is highly sensitive and can be used to determine the nucleotide sequence of a template nucleic acid which is present in only a few copies in a starting population of nucleic acids. [0034]
  • The methods and apparatuses described are generally useful for any application which the identification of any particular nucleic acid sequence is desired. For example, the methods allow for identification of single nucleotide polymorphisms (SNPs) and transcript profiling. Other uses include sequencing of artificial DNA constructs to confirm or elicit their primary sequence, or to isolate specific mutant clones from random mutagenesis screens, as well as to obtain the sequence of cDNA from single cells, whole tissues or organisms from any developmental stage or environmental circumstance in order to determine the gene expression profile from that specimen. In addition, the methods allow for the sequencing of PCR products and/or cloned DNA fragments of any size isolated from any source. [0035]
  • The methods of the present invention can be also used for the sequencing of DNA fragments generated by analytical techniques that probe higher order DNA structure by their differential sensitivity to enzymes, radiation or chemical treatment (e.g., partial DNase treatment of chromatin), or for the determination of the methylation status of DNA by comparing sequence generated from a given tissue with or without prior treatment with chemicals that convert methyl-cytosine to thymine (or other nucleotide) as the effective base recognized by the polymerase. Further, the methods of the present invention can be used to assay cellular physiology changes occurring during development or senescence at the level of primary sequence. [0036]
    • Methods of Sequencing Nucleic Acids Structure of Anchor Primers
  • Anchor primers in general include a stalk region and at least two contiguous adapter regions. The stalk region is present at the 5′ end of the anchor primer and includes a region of nucleotides for attaching the anchor primer to the solid substrate. [0037]
  • The anchor primer in general includes a region which hybridizes to a complementary sequence present in one or more members of a population of nucleic acid sequences. In some embodiments, the anchor primer includes two adjoining regions which hybridize to complementary regions ligated to separate ends of a target nucleic acid sequence. This embodiment is illustrated in FIG. 1, which is discussed in more detail below. [0038]
  • In some embodiments, the adapter regions in the anchor primers are complementary to non-contiguous regions of sequence present in a second nucleic acid sequence. Each adapter region, for example, can be homologous to each terminus of a fragment produced by digestion with one or more restriction endonucleases. The fragment can include, e.g. a sequence known or suspected to contain a sequence polymorphism. [0039]
  • In another example, the anchor primer may contain two adapter regions that are homologous to a gapped, i.e., non-contiguous because of a deletion of one or more nucleotides, region of a target nucleic acid sequence. For example, e.g., a target sequence in population of nucleic acids sequences. When adapter regions having these sequences are used, an aligning oligonucleotide corresponding to the gapped sequence may be annealed to the anchor primer along with a population of template nucleic acid molecules. [0040]
  • The anchor primer may optionally contain additional elements, e.g., one or more restriction enzyme recognition sites, RNA polymerase binding sites (e.g., a T7 promoter site). [0041]
  • One or more of the adapter regions may include, e.g., a restriction enzyme recognition site or sequences present in identified DNA sequences, e.g., sequences present in known genes. One or more adapter regions may also include sequences known to flank sequence polymorphisms. Sequence polymorphisms include nucleotide substitutions, insertions, deletions, or other rearrangements which result in a sequence difference between two otherwise identical nucleic acid sequences. An example of a sequence polymorphism is a single nucleotide polymorphism (SNP). [0042]
    • Linking of Anchor Primers to a Solid Support
  • In general, any nucleic acid capable of base-pairing can be used as an anchor primer. In some embodiments, the anchor primer is an oligonucleotide. As utilized herein the term oligonucleotide includes linear oligomers of natural or modified monomers or linkages, e.g., deoxyribonucleosides, ribonucleosides, anomeric forms thereof, peptide nucleic acids (PNAs), and the like, that are capable of specifically binding to a target polynucleotide by way of a regular pattern of monomer-to-monomer interactions. These types of interactions can include, e.g., Watson-Crick type of base-pairing, base stacking, Hoogsteen or reverse-Hoogsteen types of base-pairing, or the like. Generally, the monomers are linked by phosphodiester bonds, or analogs thereof, to form oligonucleotides ranging in size from, e.g., 3-200, 8-150, 10-100, 20-80, or 25-50 monomeric units. Whenever an oligonucleotide is represented by a sequence of letters, it is understood that the nucleotides are oriented in the 5′→3′ direction, from left-to-right, and that the letter “A” donates deoxyadenosine, the letter “T” denotes thymidine, the letter “C” denotes deoxycytosine, and the letter “G” denotes deoxyguanosine, unless otherwise noted herein. The oligonucleotides of the present invention can include non-natural nucleotide analogs. However, where, for example, processing by enzymes is required, or the like, oligonucleotides comprising naturally-occurring nucleotides are generally required for maintenance of biological function. [0043]
  • Any material can be used as the solid support material, as long as the surface allows for stable attachment of the primers and detection of nucleic acid sequences. The solid support material can be planar or can be cavitated, e.g., in a cavitated terminus of a fiber optic. In some embodiments, the solid support is optically transparent, e.g., glass. [0044]
  • The anchor primer can be linked to the solid support to reside on or within the solid support. In some embodiments, the plurality of anchor primers is linked to the solid support so they are spaced regular intervals within an array. The periodicity between primers is preferably greater than either the diffusion rate of the products of the sequencing reactions or the optical resolving power of the detection system, both of which are described in more detail below. The distance between primers on a solid substrate can be, 10-400 μm, 50-150 μm, 100-150 μm, or 150 μm. [0045]
  • An array of attachment sites on the optically transparent solid support is constructed using lithographic techniques commonly used in the construction of electronic integrated circuits as described in, e.g., techniques for attachment described in U.S. Pat. Nos. 5,5143,854, 5,445,934, 5,744,305, and 5, 800,992; Chee et al., [0046] Science 274: 610-614 (1996); Fodor et al., Nature 364: 555-556 (1993); Fodor et al., Science 251: 767-773 (1991); Gushin, et al., Anal. Biochem. 250: 203-211 (1997); Kinosita et al., Cell 93: 21-24 (1998); Kato-Yamada et al., J. Biol. Chem. 273: 19375-19377 (1998); and Yasuda et al., Cell 93: 1117-1124 (1998). Photolithography and electron beam lithography sensitize the solid support or substrate with a linking group that allows attachment of a modified biomolecule (e.g., proteins or nucleic acids). See e.g., Service, Science 283: 27-28 (1999); Rai-Choudhury, HANDBOOK OF MICROLITHOGRAPHY, MICROMACHINING, AND MICROFABRICATION, VOLUME I: MICROLITHOGRAPHY, Volume PM39, SPIE Press (1997). Alternatively, an array of sensitized sites can be generated using thin-film technology as described in Zasadzinski et al., Science 263: 1726-1733 (1994). The contents of all of these patents and publications are incorporated by reference in their entirety.
  • Anchor primers are linked to the solid substrate at the sensitized sites. A region of a solid substrate containing a linked primer is an anchor pad. Thus, by specifying the sensitized states on the solid support, it is possible to form an array or matrix of anchored pads. The anchor pads can, e.g., small diameter spots etched at evenly spaced intervals on the solid support. [0047]
  • The anchor primer can be attached to the solid support via a covalent or non-covalent interaction. Examples of such linkages common in the art include Ni[0048] 2+/hexahistidine, streptavidin/biotin, avidin/biotin, glutathione S-transferase (GST)/glutathione, monoclonal antibody/antigen, and maltose binding protein/maltose. Samples containing the appropriate tag are incubated with the sensitized substrate so that a single molecule attaches at each sensitized site.
  • The biotin-(strept-)avidin methodology provides several different ways to immobilize the anchor on the solid support. One biotin-(strept-)avidin-based anchoring method uses a thin layer of a photoactivatable biotin analog dried onto a solid surface. (Hengsakul and Cass, 1996. [0049] Biocongjugate Chem. 7: 249-254). The biotin analog is then exposed to white light through a mask, so as to create defined areas of activated biotin. Avidin (or streptavidin) is then added and allowed to bind to the activated biotin. The avidin possesses free biotin binding sites which can be utilized to “anchor” the biotinylated oligonucleotides through a biotin-(strept-)avidin linkage.
  • Alternatively, the anchor primer can be attached to the solid support with a biotin derivative possessing a photo-removable protecting group. This moiety is covalently bound to bovine serum albumin (BSA), which is attached to the solid support, e.g., a glass surface. See Pirrung and Huang, 1996. [0050] Bioconjugate Chem. 7: 317-321. A mask is then used to create activated biotin within the defined irradiated areas. Avidin may then be localized to the irradiated area, with biotinylated DNA subsequently attached through a BSA-biotin-avidin-biotin link. If desired, an intermediate layer of silane is deposited in a self-assembled monolayer on a silicon dioxide silane surface that can be patterned to localize BSA binding in defined regions. See e.g., Mooney, et al., 1996. Proc. Natl. Acad. Sci. USA 93: 12287-12291.
  • Each sensitized site on a solid support is potentially capable of attaching multiple anchor primers. Thus, each anchor pad may include one or more anchor primers. It is preferable to maximize the number of pads that have only a single productive reaction center (e.g., the number of pads that, after the extension reaction, have only a single sequence extended from the anchor primer). This can be accomplished by techniques which include, but are not limited to: (i) varying the dilution of biotinylated anchor primers that are washed over the surface; (ii) varying the incubation time that the biotinylated primers are in contact with the avidin surface; or (iii) varying the concentration of open- or closed-circular template so that, on average, only one primer on each pad is extended to generate the sequencing template. [0051]
  • In some embodiments, each individual pad contains just one linked anchor primer. Pads having only one anchor primer can be made by performing limiting dilutions of a selected anchor primer on to the solid support such that, on average, only one anchor primer is deposited on each pad. The concentration of anchor primer to be applied to a pad can be calculated utilizing, for example, a Poisson distribution model. [0052]
  • In order to maximize the number of reaction pads that contain a single anchor primer, a series of dilution experiments are performed in which a range of anchor primer concentrations or circular template concentrations are varied. For highly dilute concentrations of primers, primers and circular templates binding to the same pad will be independent of each other, and a Poisson distribution will characterize the number of anchor primers extended on any one pad. Although there will be variability in the number of primers that are actually extended, a maximum of 37% of the pads will have a single extended anchor primer (the number of pads with a single anchor oligonucleotide). This number can be obtained as follows. [0053]
  • Let N[0054] p be the average number of anchor primers on a pad and f be the probability that an anchor primer is extended with a circular template. Then the average number of extended anchor primers per pad is Npf, which is defined as the quantity a. There will be variability in the number of primers that are actually extended. In the low-concentration limit, primers and circular templates binding to the same pad will be independent of each other, and a Poisson distribution P(n) will characterize the number of anchor primers n extended on any pad. This distribution may be mathematically defined by: P(n)=( an/n!)exp(−a), with P(1)=a exp(−a). The probability P(1) assumes it maximum value exp(−1) for a=1, with 37% of pads having a single extended anchor primer.
  • A range of anchor primer concentrations and circular template concentrations may be subsequently scanned to find a value of N[0055] pf closest to 1. A preferable method to optimize this distribution is to allow multiple anchor primers on each reaction pad, but use a limiting dilution of circular template so that, on average, only one primer on each pad is extended to generate the sequencing template.
  • Alternatively, at high concentration of anchor primers, multiple anchor primers will likely be bound on each reaction pad, but a limiting dilution of circular template may be used so that, on average, only one primer on each pad anneals to a template molecule and is extended to amplify the sequencing template. [0056]
  • Where the reaction pads are arrayed on a planar surface, the individual pads are approximately 10 μm on a side, with a 100 μm spacing between adjacent pads. Hence, on a 1 cm surface a total of approximately 10,000 pads could be deposited, and, according to the Poisson distribution, approximately 3700 of these will contain a single anchor primer. In certain embodiments, after the primer oligonucleotide has been attached to the solid support, modified, e.g., biotinylated, enzymes are deposited to bind to the remaining, unused avidin binding sites on the planar surface. [0057]
  • In other embodiments multiple anchor primers are attached to any one individual pad in an array. Limiting dilutions of a plurality of circular nucleic acid templates (described in more detail below) may be hybridized to the anchor primers so immobilized such that, on average, only one primer on each pad is hybridized to a nucleic acid template. Library concentrations to be used may be calculated utilizing, for example, limiting dilutions and a Poisson distribution model. [0058]
    • Libraries of Single-stranded Circular Templates
  • A plurality of nucleic acid templates, e.g., a nucleic acid library, in general includes open circular or closed circular nucleic acid molecules. A “closed circle” is a covalently closed circular nucleic acid molecule, e.g., a circular DNA or RNA molecule. An “open circle” is a linear single-stranded nucleic acid molecule having a 5′ phosphate group and a 3′ hydroxyl group. The ends of a given open circle nucleic acid molecule can be ligated by DNA ligase. Sequences at the 5′ and 3′ ends of the open circle molecule are complementary to two regions of adjacent nucleotides in a second nucleic acid molecule, e.g., an adapter region of an anchor primer, or to two regions that are nearly adjoining in a second DNA molecule. Thus, the ends of the open-circle molecule can be ligated using DNA ligase, or extended by DNA polymerase in a gap-filling reaction. Open circles are described in detail in Lizardi, U.S. Pat. No. 5,854,033. An open circle can be converted to a closed circle in the presence of a DNA ligase (for DNA) or RNA ligase following, e.g., annealing of the open circle to an anchor primer. [0059]
  • Circularized oligonucleotide probes (i e., padlock probes) are comprised of two target sequence-complementarity sequences which are connected by a linker which may possess detectable functionalities. The linkers can be ligated to ends of members of a library of nucleic acid sequences that have been, e.g., physically sheared or digested with restriction endonucleases. [0060]
  • The 5′- and 3′-terminal regions of these linear oligonucleotides are designed to basepair adjacent to one another on a specific target sequence strand, thus the termini of the linear oligonucleotide are brought into juxtaposition by hybridization to the target sequence. This juxtaposition allows the two probe segments (if properly hybridized) to be covalently-bound by enzymatic ligation (e.g., with T[0061] 4 DNA ligase), thus converting the probes to circularly-closed molecules which are catenated to the specific target sequences (see e.g., Nilsson, et al., 1994. Science 265: 2085-2088). The resulting probes are suitable for the simultaneous analysis of many gene sequences both due to their specificity and selectivity for gene sequence variants (see e.g., Lizardi, et al., 1998. Nat. Genet. 19: 225-232; Nilsson, et al., 1997. Nat. Genet. 16: 252-255) and due to the fact that the resulting reaction products remain localized to the specific target sequences. Moreover, intramolecular ligation of many different probes is expected to be less susceptible to non-specific cross-reactivity than multiplex PCR-based methodologies where non-cognate pairs of primers can give rise to irrelevant amplification products (see e.g., Landegren and Nilsson, 1997. Ann. Med. 29: 585-590).
  • The starting library can be either single-stranded or double-stranded, as long as it includes a region that, if present in the library, is available for annealing, or can be made available for annealing, to an anchor primer sequence. [0062]
  • Library templates can include multiple elements, including, but not limited to, one or more regions that are complementary to the anchor primer. For example, the template libraries may include a region complementary to a sequencing primer, a control nucleotide region, and an insert sequence comprised of the sequencing template to be subsequently characterized. As is explained in more detail below, the control nucleotide region is used to calibrate the relationship between the amount of byproduct and the number of nucleotides incorporated. As utilized herein the term “complement” refers to nucleotide sequences that are able to hybridize to a specific nucleotide sequence to form a matched duplex. [0063]
  • In one embodiment, a library template includes: (i) two distinct regions that are complementary to the anchor primer, (ii) one region complementary to the sequencing primer, (iii) one control nucleotide region, (iv) an insert sequence of 30-100 nucleotides that is to be sequenced. The template can, of course, include two, three, or all four of these features. [0064]
  • The template nucleic acid can be constructed from any source of nucleic acid, e.g., any cell, tissue, or organism, and can be generated by any art-recognized method. Suitable methods include, e.g., sonication of genomic DNA and digestion with one or more restriction endonucleases (RE) to fragment a population of nuclei acid molecules, e.g., genomic DNA. Preferably, one or more of the restriction enzymes have distinct four-base recognition sequences. Examples of such enzymes include, e.g., Sau3AI, MspI, and TaqI. Preferably, the enzymes are used in conjunction with anchor primers having regions containing recognition sequences for the corresponding restriction enzymes. In some embodiments, the one or both adapter regions anchor primers contain additional sequences adjoining known restriction enzyme recognition sequences, thereby allowing for capture or annealing of specific restriction fragments of interest to the anchor primer. [0065]
  • In other embodiments, the restriction enzyme is used with a type IIS restriction enzyme. [0066]
  • Alternatively, template libraries can be made by generating a complementary DNA (cDNA) library from RNA, e.g., messenger RNA (mRNA). The cDNA library can, if desired, be further processed with restriction endonucleases to obtain either 3′ signature sequences, internal fragments, or 5′ fragments. adapter regions in the anchor primer, libraries containing a sequence of interest, e.g., a known or suspected sequence polymorphism on a restriction fragment. [0067]
    • Annealing and Amplification of Primer-Template Nucleic Acid Complexes
  • Libraries of nucleic acids are annealed to anchor primer sequences using recognized techniques (see, e.g., Hatch, et al., 1999. [0068] Genet. Anal. Biomol. Engineer. 15: 35-40; Kool, U.S. Pat. No.5,714, 320 and Lizardi, U.S. Pat. No.5,854,033). In general, any procedure for annealing the anchor primers to the template nucleic acid sequences is suitable as long as it results in formation of specific, i.e., perfect or nearly perfect, complementarity between the adapter region or regions in the anchor primer sequence and a sequence present in the template library.
  • A number of in vitro nucleic acid amplification techniques may be utilized to extend the anchor primer sequence. The size of the amplified DNA should be smaller than the size of the anchor pad and also smaller than the distance between anchoring pads. [0069]
  • The amplification is typically performed in the presence of a polymerase, e.g., a DNA or RNA-directed DNA polymerase, and one, two, three, or four types of nucleotide triphosphates, and, optionally, auxiliary binding proteins. In general, any polymerase capable of extending a primed 3′-OH group can be used a long as it lacks a 3′ to 5′ exonuclease activity. Suitable polymerases include, e.g., the DNA polymerases from [0070] Bacillus stearothermophilus, Thermus acquaticus, Pyrococcus furiosis, Thermococcus litoralis, and Thermus thermophilus, bacteriophage T4 and T7, and the E. coli DNA polymerase I Klenow fragment. Suitable RNA-directed DNA polymerases include, e.g., the reverse transcriptase from the Avian Myeloblastosis Virus, the reverse transcriptase from the Moloney Murine Leukemia Virus, and the reverse transcriptase from the Human Immunodeficiency Virus-I.
  • A number of in vitro nucleic acid amplification techniques have been described. These amplification methodologies may be differentiated into those methods: (i) which require temperature cycling—polymerase chain reaction (PCR) (see e.g., Saiki, et al., 1995. [0071] Science 230: 1350-1354), ligase chain reaction (see e.g., Barany, 1991. Proc. Natl. Acad. Sci. USA 88: 189-193; Barringer, et al., 1990. Gene 89: 117-122) and transcription-based amplification (see e.g., Kwoh, et al., 1989. Proc. Natl. Acad. Sci. USA 86: 1173-1177) and (ii) isothermal amplification systems—self-sustaining, sequence replication (see e.g., Guatelli, et al., 1990. Proc. Natl. Acad. Sci. USA 87: 1874-1878); the Qβ replicase system (see e.g., Lizardi, et al., 1988. BioTechnology 6: 1197-1202); strand displacement amplification Nucleic Acids Res. 1992 April 11;20(7):1691-6.; and the methods described in PNAS 1992 January 1;89(1):392-6; and NASBA J Virol Methods. 1991 Dec;35(3):273-86.
  • Isothermal amplificaion also includes rolling circle-based amplification (RCA). RCA is discussed in, e.g., Kool, U.S. Pat. No. 5,714,320 and Lizardi, U.S. Pat. No. 5,854,033; Hatch, et al., 1999. [0072] Genet. Anal. Biomol. Engineer. 15: 35-40. The result of the RCA is a single DNA strand extended from the 3′ terminus of the anchor primer (and thus is linked to the solid support matrix) and including a concatamer containing multiple copies of the circular template annealed to a primer sequence. Typically, 10,000 or more copies of circular templates, each having a size of approximately 100 nucleotides size range, can be obtained with RCA.
  • The product of RCA amplification following annealing of a circular nucleic acid molecule to an anchor primer is shown schematically in FIG. 1. A circular template [0073] nucleic acid 102 is annealed to an anchor primer 104, which has been linked to a surface 106 at its 5′ end and has a free 3′ OH available for extension. The circular template nucleic acid 102 includes two adapter regions 108 and 110 which are homologous to regions of sequence in the anchor primer 104. Also included in the circular template nucleic acid 102 is an insert 112 and a region 114 homologous to a sequencing primer, which is used in the sequencing reactions described below.
  • Upon annealing, the free 3′-OH on the [0074] anchor primer 104 can be extended using sequences within the template nucleic acid 102. The anchor primer 102 can be extended along the template multiple times, with each iteration adding to the sequence extended from the anchor primer a sequence complementary to the circular template nucleic acid. Four iterations, or four rounds of rolling circle replication, are shown in FIG. 1 as the extended anchor primer amplification product 114. Extension of the anchor primer results in an amplification product covalently attached to the substrate 106.
  • Circular oligonucleotides which are generated during polymerase-mediated DNA replication are dependent upon the relationship between the template and the site of replication initiation. In double-stranded DNA templates, the critical features include whether the template is linear or circular in nature, and whether the site of initiation of replication (i.e., the replication “fork”) is engaged in synthesizing both strands of DNA or only one. In conventional double-stranded DNA replication, the replication fork is treated as the site at which the new strands of DNA are synthesized. However, in linear molecules (whether replicated unidirectionally or bidirectionally), the movement of the replication fork(s) generate a specific type of structural motif. If the template is circular, one possible spatial orientation of the replicating molecule takes the form of an θ structure. [0075]
  • Alternatively, RCA can occur when the replication of the duplex molecule begins at the origin. Subsequently, a nick opens one of the strands, and the free 3′-terminal hydroxyl moiety generated by the nick is extended by the action of DNA polymerase. The newly synthesized strand eventually displaces the original parental DNA strand. This aforementioned type of replication is known as rolling-circle replication (RCR) because the point of replication may be envisaged as “rolling around” the circular template strand and, theoretically, it could continue to do so indefinitely. As it progresses, the replication fork extends the outer DNA strand the previous partner. Additionally, because the newly synthesized DNA strand is covalently-bound to the original template, the displaced strand possesses the original genomic sequence (e.g., gene or other sequence of interest) at its 5′-terminus. In rolling-circle replication, the original genomic sequence is followed by any number of “replication units” complementary to the original template sequence, wherein each replication unit is synthesized by continuing revolutions of said original template sequence. Hence, each subsequent revolution displaces the DNA which is synthesized in the previous replication cycle. [0076]
  • In vivo, rolling-circle replication is utilized in several biological systems. For example, in certain bacteriophage, their genome consists of single-stranded, circular DNA. During replication, the circular DNA is initially converted to a duplex form, which is then replicated by the aforementioned rolling-circle replication mechanism. The displaced terminus generates a series of genomic units, which can be cleaved and inserted into the phage particles, or they can be utilized for further replication cycles by the phage. Additionally, the displaced single-strand of a rolling-circle can be converted to duplex DNA by synthesis of a complementary DNA strand. This synthesis can be used to generate the concatemeric duplex molecules required for the maturation of certain phage DNAs. For example, this provides the principle pathway by which λ bacteriophage matures. Rolling-circle replication is also used in vivo to generate amplified rDNA in Xenopus oocytes, and this fact may help explain why the amplified rDNA is comprised of a large number of identical repeating units. In this case, a single genomic repeating unit is converted into a rolling-circle. The displaced terminus is then converted into duplex DNA which is subsequently cleaved from the circle so that the two termini can be ligated together so as to generate the amplified circle of rDNA. [0077]
  • Through the use of the RCR reaction, a strand may be generated which represents many tandem copies of the complement to the circularized molecule. For example, RCR has recently been utilized to obtain an isothermal cascade amplification reaction of circularized padlock probes in vitro in order to detect single-copy genes in human genomic DNA samples (see Lizardi, et al., 1998. [0078] Nat. Genet. 19: 225-232). In addition, RCR has also been utilized to detect single DNA molecules in a solid phase-based assay, although difficulties arose when this technique was applied to in situ hybridization (see Lizardi, et al., 1998. Nat. Genet. 19: 225-232).
  • The development of a method of amplifying short DNA molecules which have immobilized to a solid support, termed rolling circle amplification (RCA) has been recently described in the literature (see e.g., Hatch, et al., 1999. Rolling circle amplification of DNA immobilized on solid surfaces and its application to multiplex mutation detection. [0079] Genet. Anal. Biomol. Engineer. 15: 35-40; Zhang, et al., 1998. Amplification of target-specific, ligation-dependent circular probe. Gene 211: 277-85; Baner, et al., 1998. Signal amplification of padlock probes by rolling circle replication. Nucl. Acids Res. 26: 5073-5078; Liu, et al., 1995. Rolling circle DNA synthesis: small circular oligonucleotides as efficient templates for DNA polymerase. J. Am. Chem. Soc. 118: 1587-1594; Fire and Xu, 1995. Rolling replication of short DNA circles. Proc. Natl. Acad. Sci. USA 92: 4641-4645; Nilsson, et al., 1994. Padlock probes: circularizing oligonucleotides for localized DNA detection. Science 265: 2085-2088). RCA targets specific DNA sequences through hybridization and a DNA ligase reaction. The circular product is then subsequently used as a template in a rolling circle replication reaction.
  • Rolling-circle amplification (RCA) driven by DNA polymerase can replicate circularized oligonucleotide probes with either linear or geometric kinetics under isothermal conditions. In the presence of two primers (one hybridizing to the +strand, and the other, to the −strand of DNA), a complex pattern of DNA strand displacement ensues which possesses the ability to generate 1×10[0080] 9 or more copies of each circle in a short period of time (i.e., less-than 90 minutes), enabling the detection of single-point mutations within the human genome. Using a single primer, RCA generates hundreds of randomly-linked copies of a covalently closed circle in several minutes. If solid support matrix-associated, the DNA product remains bound at the site of synthesis, where it may be labeled, condensed and imaged as a point light source. For example, linear oligonucleotide probes, which can generate RCA signals, have been bound covalently onto a glass surface. The color of the signal generated by these probes indicates the allele status of the target, depending upon the outcome of specific, target-directed ligation events. As RCA permits millions of individual probe molecules to be counted and sorted, it is particularly amenable for the analysis of rare somatic mutations. RCA also shows promise for the detection of padlock probes bound to single-copy genes in cytological preparations.
  • In addition, a solid-phase RCA methodology has also been developed to provide an effective method of detecting constituents within a solution. Initially, a recognition step is used to generate a complex consisting of a DNA primer duplexed with a circular template is bound to a surface. A polymerase enzyme is then used to amplify the bound complex. RCA uses small DNA probes that are amplified to provide an intense signal using detection methods, including the methods described in more detail below. [0081]
  • Other examples of isothermal amplification systems include, e.g., (i) self-sustaining, sequence replication (see e.g., Guatelli, et al., 1990. [0082] Proc. Natl. Acad. Sci USA 87: 1874-1878), (ii) the Qβ replicase system (see e.g., Lizardi, et al., 1988. BioTechnology 6: 1197-1202), and (iii) nucleic acid sequence-based amplification (NASBA™; see Kievits, et al., 1991. J. Virol. Methods 35: 273-286).
    • Determining the Nucleotide Sequence of the Sequence Product
  • Amplification of a nucleic acid template as described above results in multiple copies of a template nucleic acid sequence covalently linked to an anchor primer. In one embodiment, a region of the sequence product is determined by annealing a sequencing primer to region of the template nucleic acid, and then contacting the sequencing primer with a DNA polymerase and a known nucleotide triphosphate, i.e., dATP, dCTP, dGTP, dTTP, or an analog of one of these nucleotides. [0083]
  • The sequence primer can be any length or base composition, as long as it is capable of specifically annealing to a region of the amplified nucleic acid template. No particular structure is required for the sequencing primer is required so long as it is able to specifically prime a region on the amplified template nucleic acid. Preferably, the sequencing primer is complementary to a region of the template that is between the sequence to be characterized and the sequence hybridizable to the anchor primer. The sequencing primer is extended with the DNA polymerase to form a sequence product. The extension is performed in the presence of one or more types of nucleotide triphosphates, and if desired, auxiliary binding proteins. [0084]
  • Incorporation of the dNTP is determined by assaying for the presence of a sequencing byproduct. In a preferred embodiment, the nucleotide sequence of the sequencing product is determined by measuring inorganic pyrophosphate (PPi) liberated from a nucleotide triphosphate (dNTP) as the NTP is incorporated into an extended sequence primer. This method of sequencing, termed Pyrosequencing™ technology (PyroSequencing AB, Stockholm, Sweden) can be performed in solution (liquid phase) or as a solid phase technique. PPi-based sequencing methods are described generally in, e.g., WO9813523A1, Ronaghi, et al., 1996. [0085] Anal. Biochem. 242: 84-89, and Ronaghi, et al., 1998. Science 281: 363-365 (1998). These disclosures of PPi sequencing are incorporated herein in their entirety, by reference.
  • Pyrophosphate released under these conditions can be detected enzymatically (e.g., by the generation of light in the luciferase-luciferin reaction). Such methods enable a nucleotide to be identified in a given target position, and the DNA to be sequenced simply and rapidly while avoiding the need for electrophoresis and the use of potentially dangerous radiolabels. [0086]
  • PPi can be detected by a number of different methodologies, and various enzymatic methods have been previously described (see e.g., Reeves, et al., 1969. [0087] Anal. Biochem. 28: 282-287; Guillory, et al., 1971. Anal. Biochem. 39: 170-180; Johnson, et al., 1968. Anal. Biochem. 15: 273; Cook, et al., 1978. Anal. Biochem. 91: 557-565; and Drake, et al, 1979. Anal. Biochem. 94: 117-120).
  • PPi liberated as a result of incorporation of a dNTP by a polymerase can be converted to ATP using, e.g., an ATP suflufurylase. This enzyme has been identified as being involved in sulfur metabolism. Sulfur, in both reduced and oxidized forms, is an essential mineral nutrient for plant and animal growth (see e.g., Schmidt and Jager, 1992. [0088] Ann. Rev. Plant Physiol. Plant Mol. Biol. 43: 325-349). In both plants and microorganisms, active uptake of sulfate is followed by reduction to sulfide. As sulfate has a very low oxidation/reduction potential relative to available cellular reductants, the primary step in assimilation requires its activation via an ATP-dependent reaction (see e.g., Leyh, 1993. Crit. Rev. Biochem. Mol. Biol. 28: 515-542). ATP sulfurylase (ATP: sulfate adenylyltransferase; EG 2.7.7.4) catalyzes the initial reaction in the metabolism of inorganic sulfate (SO4 −2); see e.g., Robbins and Lipmann, 1958. J. Biol. Chem. 233: 686-690; Hawes and Nicholas, 1973. Biochem. J 133: 541-550) In this reaction SO4 −2 is activated to adenosine 5′-phophosulfate (APS).
  • ATP sulfurylase has been highly purified from several sources, such as [0089] Saccharomyces cerevisiae (see e.g., Hawes and Nicholas, 1973. Biochem. J. 133: 541-550); Penicillium chrysogenum (see e.g., Renosto, et al., 1990. J. Biol. Chem. 265: 10300-10308); rat liver (see e.g., Yu, et al., 1989. Arch. Biochem. Biophys. 269: 165-174); and plants (see e.g., Shaw and Anderson, 1972. Biochem. J. 127: 237-247; Osslund, et al., 1982. Plant Physiol. 70: 39-45). Furthermore, ATP sulfurylase genes have been cloned from prokaryotes (see e.g., Leyh, et al., 1992. J. Biol. Chem. 267: 10405-10410; Schwedock and Long, 1989. Mol. Plant Microbe Interaction 2: 181-194; Laue and Nelson, 1994. J. Bacteriol. 176: 3723-3729); eukaryotes (see e.g., Cherest, et al., 1987. Mol. Gen. Genet. 210: 307-313; Mountain and Korch, 1991. Yeast 7: 873-880; Foster, et al., 1994. J. Biol. Chem. 269: 19777-19786); plants (see e.g., Leustek, et al., 1994. Plant Physiol 105: 897-90216); and animals (see e.g., Li, et al., 1995. J. Biol Chem. 270: 29453-29459). The enzyme is homo-oligomer or heterodimer, depending upon the specific source (see e.g., Leyh and Suo, 1992. J. Biol Chem. 267: 542-545).
  • ATP sulfurylase has been used for many different applications, for example, bioluminometric detection of ADP at high concentrations of ATP (see e.g., Schultz, et al., 1993. [0090] Anal. Biochem. 215: 302-304); continuous monitoring of DNA polymerase activity (see e.g., Nyrbn, 1987. Anal. Biochem. 167: 235-238); and DNA sequencing (see e.g., Ronaghi, et al., 1996. Anal. Biochem. 242: 84-89; Ronaghi, et al., 1998. Science 281: 363-365; Ronaghi, et al., 1998. Anal. Biochem. 267: 65-71).
  • Several assays have been developed for detection of the forward ATP sulfurylase reaction. The colorimetric molybdolysis assay is based on phosphate detection (see e.g., Wilson and Bandurski, 1958. [0091] J. Biol. Chem. 233: 975-981), whereas the continuous spectrophotometric molybdolysis assay is based upon the detection of NADH oxidation (see e.g., Seubert, et al., 1983. Arch. Biochem. Biophys. 225: 679-691; Seubert, et al., 1985. Arch. Biochem. Biophys. 240: 509-523). The later assay requires the presence of several detection enzymes. In addition, several radioactive assays have also been described in the literature (see e.g., Daley, et al., 1986. Anal. Biochem. 157: 385-395). For example, one assay is based upon the detection of 32PPi released from 32P-labeled ATP (see e.g., Seubert, et al., 1985. Arch. Biochem. Biophys. 240: 509-523) and another on the incorporation of 35S into [35S]-labeled APS (this assay also requires purified APS kinase as a coupling enzyme; see e.g., Seubert, et al., 1983. Arch. Biochem. Biophys. 225: 679-691); and a third reaction depends upon the release of 35SO4 −2 from [35S]-labeled APS (see e.g., Daley, et al, 1986. Anal. Biochem. 157: 385-395).
  • For detection of the reversed ATP sulfurylase reaction a continuous spectrophotometric assay (see e.g., Segel, et al., 1987. [0092] Methods Enzymol. 143: 334-349); a bioluminometric assay (see e.g., Balharry and Nicholas, 1971. Anal. Biochem. 40: 1-17); an 35SO4 −2 release assay (see e.g., Seubert, et al., 1985. Arch. Biochem. Biophys. 240: 509-523); and a 32PPi incorporation assay (see e.g., Osslund, et al., 1982. Plant Physiol. 70: 39-45) have been previously described.
  • ATP produced by an ATP sulfurylase can be converted using enzymatic reactions which convert ATP to light. Light-emitting chemical reactions (i.e., chemiluminescence) and biological reactions (i.e., bioluminescence) are widely used in analytical biochemistry for sensitive measurements of various metabolites. In bioluminescent reactions, the chemical reaction that leads to the emission of light is enzyme-catalyzed. For example, the luciferin-luciferase system allows for specific assay of ATP and the bacterial luciferase-oxidoreductase system can be used for monitoring of NAD(P)H. Both systems have been extended to the analysis of numerous substances by means of coupled reactions involving the production or utilization of ATP or NAD(P)H (see e.g., Kricka, 1991. Chemiluminescent and bioluminescent techniques. [0093] Clin. Chem. 37: 1472-1281).
  • The development of new reagents have made it possible to obtain stable light emission proportional to the concentrations of ATP (see e.g., Lundin, 1982. Applications of firefly luciferase In; [0094] Luminescent Assays (Raven Press, New York) or NAD(P)H (see e.g., Lovgren, et al., Continuous monitoring of NADH-converting reactions by bacterial luminescence. J. Appl. Biochem. 4: 103-111). With such stable light emission reagent, it is possible to make endpoint assays and to calibrate each individual assay by addition of a known amount of ATP or NAD(P)H. In addition, a stable light-emitting system also allows continuous monitoring of ATP- or NAD(P)H-converting systems.
  • Suitable enzymes for converting ATP into light include luciferases, e.g., insect luciferases. Luciferases produce light as an end-product of catalysis. The best known light-emitting enzyme is that of the firefly, [0095] Photinus pyralis (Coleoptera). The corresponding gene has been cloned and expressed in bacteria (see e.g., de Wet, et al., 1985. Proc. Natl. Acad. Sci. USA 80: 7870-7873) and plants (see e.g., Ow, et al., 1986. Science 234: 856-859), as well as in insect (see e.g., Jha, et al., 1990. FEBS Lett. 274: 24-26) and mammalian cells (see e.g., de Wet, et al., 1987. Mol. Cell. Biol. 7: 725-7373; Keller, et al, 1987. Proc. Natl. Acad. Sci. USA 82: 3264-3268). In addition, a number of luciferase genes from the Jamaican click beetle, Pyroplorus plagiophihalamus (Coleoptera), have recently been cloned and partially characterized (see e.g., Wood, et al., 1989. J. Biolumin. Chemilumin. 4: 289-301; Wood, et al., 1989. Science 244: 700-702). Distinct luciferases can sometimes produce light of different wavelengths, which may enable simultaneous monitoring of light emissions at different wavelengths. Accordingly, these aforementioned characteristics are unique, and add new dimensions with respect to the utilization of current reporter systems.
  • Firefly luciferase catalyzes bioluminescence in the presence of luciferin, [0096] adenosine 5′-triphosphate (ATP), magnesium ions, and oxygen, resulting in a quantum yield of 0.88 (see e.g., McElroy and Selinger, 1960. Arch. Biochem. Biophys. 88: 136-145). The firefly luciferase bioluminescent reaction can be utilized as an assay for the detection of ATP with a detection limit of approximately 1×10−13 M (see e.g., Leach, 1981. J. Appl. Biochem. 3: 473-517). In addition, the overall degree of sensitivity and convenience of the luciferase-mediated detection systems have created considerable interest in the development of firefly luciferase-based biosensors (see e.g., Green and Kricka, 1984. Talanta 31: 173-176; Blum, et al., 1989. J. Biolumin. Chemilumin. 4: 543-550).
  • Using the above-described enzymes, the sequence primer is exposed to a polymerase and a known dNTP. If the dNTP is incorporated onto the 3′ end of the primer sequence, the dNTP is cleaved and a PPi molecule is liberated. The PPi is then converted to ATP with ATP sulfurylase. Preferably, the ATP sulfurylase is present at a sufficiently high concentration that the conversion of PPi proceeds with first-order kinetics. In the presence of luciferase, the ATP is hydrolyzed to liberate a photon. The reaction preferably has a sufficient concentration of luciferase present within the reaction mixture such that the reaction, ATP→ADP+PO[0097] 4 3−+ photon (light), proceeds with first-order kinetics. The photon can be measured using methods and apparatuses described below.
  • **can be detected by examining a relative increase in sequencing by [0098]
  • For most applications it is desirable to wash away diffusible sequencing reagents, e.g., unincorporated dNTPs, with a wash buffer. Any wash buffer used in pyrophosphate sequencing can be used. An example of a wash buffer is 10 mM Trisc-HCl (pH 7.5), 1 mM EDTA, 2 M NaCl, 1% Tween 20 (Nyren et al., Anal. Biochem. 208:171-75, 1993). [0099]
  • In some embodiments, the concentration of reactants in the sequencing reaction include 1 pmol DNA, 3 pmol polymerase, 40 pmol dNTP in 0.2 ml buffer. See Ronaghi, et al., [0100] Anal. Biochem. 242: 84-89 (1996).
  • The sequencing reaction can be performed with each of four predetermined nucleotides, if desired. A “complete” cycle generally includes sequentially administering sequencing reagents for each of the nucleotides dATP, dGTP, dCTP and dTTP (or dUTP), in a predetermined order. Unincorporated dNTPs are washed away between each of the nucleotide additions. Alternatively, unincorporated dNTPs are degraded by apyrase (see below). The cycle is repeated as desired until the desired amount of sequence of the sequence product is obtained. In some embodiments, about 10-1000, 10-100, 10-75, 20-50, or about 30 nucleotides of sequence information is obtained from one annealed primer. [0101]
  • Luciferase can hydrolyze dATP directly with concomitant release of a photon. This results in a false positive signal because the hydrolysis occurs independent of incorporation of the dATP into the extended sequencing primer. To avoid this problem, a dATP analog can be used which is incorporated into DNA, i e., it is a substrate for a DNA polymerase, but is not a substrate for luciferase. One such analog is α-thio-ATP such as [0102] adenosine 5′-phosphosulfate (APS). Thus, use of APS avoids the spurious photon generation that can occur when dATP is hydrolyzed without being incorporated into a growing nucleic acid chain.
  • Typically, the PPi-based detection is calibrated by the measurement of the light released following the addition of control nucleotides to the sequencing reaction mixture immediately after the addition of the sequencing primer. This allows for normalization of the reaction conditions. Incorporation of two or more identical nucleotides in succession is revealed by a corresponding increase in the amount of light released. Thus, a two-fold increase in released light relative to control nucleotides reveals the incorporation of two successive dNTPs into the extended primer. [0103]
  • If desired, apyrase may be “washed” or “flowed” over the surface of the solid support so as to facilitate the degradation of any remaining, non-incorporated dNTPs within the sequencing reaction mixture. Upon treatment with apyrase, any remaining reactants are washed away in preparation for the following dNTP incubation and photon detection steps. Alternatively, the apyrase may be bound to the solid support. [0104]
  • When the support is planar, the pyrophosphate sequencing reactions preferably take place in a thin, aqueous reaction chamber comprising an optically-transparent solid support surface and an optically-transparent cover. Sequencing reagents may then be delivered by flowing them across the surface of the substrate. When the support is not planar, the reagents may be delivered by dipping the solid support into baths of any given reagents. [0105]
  • In some embodiments, the enzymes utilized in the pyrophosphate sequencing reaction (e.g., sulfurylase, luciferase, and apyrase) may be immobilized onto the solid support. When luciferase is immobilized, it is preferably less than 50 μm from an anchored primer. [0106]
  • The photons generated by luciferase may be quantitated using a variety of detection apparatuses, e.g., a photomultiplier tube, charge-coupled display (CCD), absorbance photometer, and a luminometer, as well as the apparatuses described herein. In a preferred embodiment, the quantitation of the emitted photons is accomplished by the use of charge-coupled display (CCD) camera fitted with a microchannel plate intensifier. CCD detectors are described in, e.g., Bronks, et al., 1995. [0107] Anal Chem. 65: 2750-2757. Preferably, the CCD camera uses a custom designed and fabricated CCD possessing a total of 16 million pixels (i.e., 4,000×4,000 pixel array) which can detect approximately 1% of the photons produced and can convert 40% to 80% of the photons produced into an actual measurable signal. With this system, approximately 1% of the photons produced are detected. This system can convert 40% to 80% of the photons produced into an actual measurable signal. Additionally, this CCD system possesses a minimum signal-to-noise ratio of 5:1, with a 10:1 signal-to-noise ratio being preferable.
    • Apparatuses for Sequencing Nucleic Acids
  • Also provided in the invention are apparatuses for sequencing nucleic acids. In some embodiments, the apparatuses include anchor primers attached to planar substrates. Nucleic acid sequence information can be detected using conventional optics or fiber-optic based systems attached to the planar substrate. In other embodiments, the apparatuses include anchor primers attached to the termini of fiber-optic arrays. In these embodiments, sequence information can be obtained directly from the termini of the fiber optic array. [0108]
    • Apparatus for Sequencing Nucleic Acids
  • An apparatus for sequencing nucleic acids is illustrated in FIG. 2. The apparatus includes an [0109] inlet conduit 200 in communication with a detachable perfusion chamber 220. The inlet conduit 200 allows for entry of sequencing reagents via a plurality of tubes 202-212, which are each in communication with a plurality of sequencing dispensing reagent vessels 214-224.
  • Reagents are introduced through the [0110] conduit 200 into the perfusion chamber 220 using either a pressurized system or pumps to drive positive flow. Typically, the reagent flow rates are from 1 to 50 ml/minute with volumes from 0.100 ml to continuous flow (for washing). Valves are under computer control to allow cycling of nucleotides and wash reagents. Sequencing reagents, e.g., polymerase can be either pre-mixed with nucleotides or added in stream. A manifold brings all six tubes 202-212 together into one for feeding the perfusion chamber. Thus several reagent delivery ports allow access to the perfusion chamber. For example, one of the ports may be utilized to allow the input of the aqueous sequencing reagents, while another port allows these reagents (and any reaction products) to be withdrawn from the perfusion chamber.
  • The [0111] perfusion chamber 200 contains a substrate to which a plurality of anchor primers have been attached. This can be a planar substrate containing one or more anchored primers in anchor pads formed at the termini of a bundled fiber optic arrays. The latter substrate surface is discussed in more detail below.
  • The perfusion chamber allows for a uniform, linear flow of the required sequencing reagents, in aqueous solution form, over the amplified nucleic acids and allows for the rapid and complete exchange of these reagents. Thus, it is suitable for performing pyrophosphate-based sequencing reaction. The perfusion chamber can also be used to prepare the anchor primers and perform amplification reactions, e.g., the RCA reactions described herein. [0112]
  • The perfusion chamber is linked to an [0113] imaging system 230, which includes a CCD system in association with conventional optics or a fiber optic bundle. For DNA immobilized on an anchor pad of 10 μm in diameter, a 100 μm diameter lens for CCD imaging is preferably placed 1 cm above the slide. For fiber-optic based imaging, it is preferable to incorporate the optical fibers directly into the cover slip.
  • The [0114] imaging system 230 is used to collect light from the reactors on the substrate surface. Light can be imaged, for example, onto a CCD using a high sensitivity low noise apparatus known in the art.
  • The imaging system is linked to a computer control and [0115] data collection system 240. In general, any commonly available hardware and software package can be used. The computer control and data collection system is also linked to the conduit 200 to control reagent delivery.
  • An example of a perfusion chamber of the present invention is illustrated in FIG. 3. The perfusion chamber includes a sealed compartment with transparent upper and lower slide. It is designed to allow linear flow of solution over the surface of the substrate surface and to allow for fast exchange of reagents. Thus, it is suitable for carrying out, for example, the pyrophosphate sequencing reactions. Laminar flow across the perfusion chamber can be optimized by decreasing the width and increasing the length of the chamber. [0116]
  • The perfusion chamber is preferably detached form the imaging system while it is being prepared and only placed on the imaging system when sequencing analyses is performed. [0117]
  • In one embodiment, the solid support (i.e., a DNA chip or glass slide) is held in place by a metal or plastic housing, which may be assembled and disassembled to allow replacement of said solid support. [0118]
  • The lower side of the solid support of the perfusion chamber carries the reaction center array and, with a traditional optical-based focal system, a high numerical aperture objective lens is used to focus the image of the reaction center array onto the CCD imaging system. [0119]
  • The photons generated by the pyrophosphate sequencing reaction are captured by the CCD only if they pass through a focusing device (e.g., an optical lens or optical fiber) and are focused upon a CCD element. However, the emitted photons should escape equally in all directions. In order to maximize their subsequent “capture” and quantitation when utilizing a planar array (e.g., a DNA chip), it is preferable to collect the photons immediately at the planar solid support (e.g., the cover slip). This is accomplished by either: (i) utilizing optical immersion oil between the cover slip and a traditional optical lens or optical fiber bundle or, preferably, (ii) incorporating optical fibers directly into the cover slip itself. Similarly, when a thin, optically-transparent planar surface is used, the optical fiber bundle can also be placed against its back surface, eliminating the need to “image” through the depth of the entire reaction/perfusion chamber. [0120]
  • In some embodiments, the solid support is coupled to a bundle of optical fibers, which are used to detect and transmit sequence reaction of byproducts. The total number of optical fibers within the bundle may be varied so as to match the number of individual arrays utilized in the sequencing reaction. The number of optical fibers incorporated into the bundle is designed to match the CCD (i.e., approximately 30 mm×30mm) so as to allow 1:1 imaging. Commercially-available optical fiber bundles range from 25 cm×25 cm to 10 μm in diameter. The desired number of optical fibers are initially fused into a bundle, the terminus of which is cut and polished so as to form a “wafer” of the required thickness (e.g., 1.5 mm). The resulting optical fiber wafers possess similar handling properties to that of a plane of glass. The individual fibers can be any size diameter (e.g., 3 μm to 100 μm). [0121]
    • Fiber Optic Substrate Arrays With Linked Anchor Primers
  • In other embodiments, the planar support is omitted and the anchor primers are linked directly to the termini of the optical fibers. Preferably, the anchor primers are attached to termini that are cavitated as shown schematically in FIG. 4. The termini are treated, e.g., with acid, to form a hemispherical shape indentation, or cavitation, that ranges from approximately one-half the depth of an individual optical fiber up to 2- to 3-times the diameter of the fiber. When used for pyrophosphate-based sequencing, the cavity is preferably 50 μm deep. [0122]
  • Cavities can be introduced into the termini of the fibers by placing one side of the optical fiber wafer into an acid bath for a variable amount of time. The amount of time can vary depending upon the overall depth of the reaction cavity desired (see e.g., Walt, et al., 1996. [0123] Anal. Chem. 70: 1888). Several methods are known in the art for attaching molecules (and detecting the attached molecules) in the cavities etched in the ends of fiber optic bundles. See, e.g., Michael, et al., Anal. Chem. 70: 1242-1248 (1998); Ferguson, et al., Nature Biotechnology 14: 1681-1684 (1996); Healey and Walt, Anal. Chem. 69: 2213-2216 (1997). A pattern of reactive sites can also be created in the microwell, using photolithographic techniques similar to those used in the generation of a pattern of reaction pads on a planar support. See, Healey, et al., Science 269: 1078-1080 (1995); Munkholm and Walt, Anal. Chem. 58: 1427-1430 (1986), and Bronk, et al., Anal. Chem. 67: 2750-2757 (1995).
  • The opposing side of the optical fiber wafer (i.e., the non-etched side) is highly polished so as to allow optical-coupling (e.g., by immersion oil or other optical coupling fluids) to a second, optical fiber bundle. This second optical fiber bundle exactly matches the diameter of the optical wafer containing the reaction chambers, and serve to act as a conduit for the transmission of the photons, generated by the pyrophosphate sequencing reaction, to its attached CCD imaging system or camera. [0124]
  • In a preferred embodiment, the individual optical fibers utilized to generate the fused optical fiber bundle/wafer are larger in diameter (i.e., 6 μm to 12 μm) than those utilized in the optical imaging system (i.e., 3 μm). Thus, several of the optical imaging fibers can be utilized to image a single reaction site. [0125]
  • The etched, hemispherical geometry allows for simultaneously reducing background signal from the PP[0126] i released from adjacent anchor pads. In contrast to use of a “chip”-based geometry, wherein the required sequencing reagents are “flowed” over the surface of the solid support matrix (i.e., the anchor pads), delivery of the various sequencing reagents in acid-etched optical fiber bundle embodiment is performed by immersion of the acid-etched cavities, alternately, into dNTP/APS/sulfurylase reagents and then, subsequently, into the apyrase reagents to facilitate the degradation of any remaining dNTPs.
  • It has been unexpectedly found that this system is markedly more efficient than the currently-utilized CCD capture techniques. For a hemispherical-shaped acid-etched cavity, approximately 85% of the emitted photons will impinge upon, and be directed down the length of the individual optical fiber to the CCD camera. Thus, fewer numbers of rolling circle amplification reactions are required to generate a detectable signal. [0127]
    • Mathematical Analysis Underlying Optimization of the Pyrophosphate Sequencing Reaction
  • While not wishing to be bound by theory, it is believed that optimization of reaction conditions can be optimized using assumptions underlying the following analyses. [0128]
  • Solid-phase pyrophosphate sequencing was initially developed by combining a solid-phase technology and a sequencing-by-synthesis technique utilizing bioluminescence (see e.g., Ronaghi, et al., 1996. Real-time DNA sequencing using detection of pyrophosphate release. [0129] Anal. Biochem. 242: 84-89). In the solid-phase methodology, an immobilized, primed DNA strand is incubated with DNA polymerase, ATP sulfurylase, and luciferase. By stepwise nucleotide addition with intermediate washing, the event of sequential polymerization can be followed. A remarkable increase in signal-to-noise ratio was obtained by the use of α-thio dATP in the system. This DATP analog is demonstrated to be efficiently incorporated by DNA polymerase while being silent for luciferase, allowing the sequencing reaction to be performed in real-time. In these early studies, sequencing of a PCR product using streptavidin-coated magnetic beads as a solid support was presented. However, it was found that the loss of the beads during washing, which was performed between each nucleotide and enzyme addition, was the limiting factor to sequence longer stretches.
  • Currently, pyrophosphate sequencing methodologies have a reasonably well-established history for ascertaining the DNA sequence from many identical copies of a single DNA sequencing template (see e.g., Ronaghi, et al., 1996. Real-Time DNA Sequencing Using Detection of Pyrophosphate Release, [0130] Anal. Biochem. 242: 84-89; Nyrén, et al., Method of Sequencing DNA, patent WO9813523A1 (issued Apr. 2, 1998; filed Sep. 26, 1997); Ronaghi, et al., 1998. A Sequencing Method Based on Real-Time Pyrophosphate Science 281: 363-365 (1998). Pyrophosphate (PPi)-producing reactions can be monitored by a very sensitive technique based on bioluminescence (see e.g., Nyrén, et al., 1996. pp. 466-496 (Proc. 9th Inter. Symp. Biolumin. Chemilumin.). These bioluminometric assays rely upon the detection of the PPi released in the different nucleic acid-modifying reactions. In these assays, the PPi which is generated is subsequently converted to ATP by ATP sulfurylase and the ATP production is continuously monitored by luciferase. For example, in polymerase-mediated reactions, the PPi is generated when a nucleotide is incorporated into a growing nucleic acid chain being synthesized by the polymerase. While generally, a DNA polymerase is utilized to generate PPi during a pyrophosphate sequencing reaction (see e.g., Ronaghi, et al, 1998. Doctoral Dissertation, The Royal Institute of Technology, Dept. of Biochemistry (Stockholm, Sweden)), it is also possible to use reverse transcriptase (see e.g., Karamohamamed, et al., 1996. pp. 319-329 (Proc. 9th Inter. Symp. Biolumin. Chemilumin.) or RNA polymerase (see e.g., Karamohamamed, et al., 1998. BioTechniques 24: 302-306) to follow the polymerization event.
  • For example, a bioluminometric primer extension assay has been utilized to examine single nucleotide mismatches at the 3′-terminus (see e.g., Nyrén, et al., 1997. [0131] Anal. Biochem. 244: 367-373). A phage promoter is typically attached onto at least one of the arbitrary primers and, following amplification, a transcriptional unit may be obtained which can then be subjected to stepwise extension by RNA polymerase. The transcription-mediated PPi-release can then be detected by a bioluminometric assays (e.g., ATP sulfurylase-luciferase). By using this strategy, it is likely to be possible to sequence double-stranded DNA without any additional specific sequencing primer. In a series of “run-off” assays, the extension by T7 phage RNA polymerase has been examined and was found to be rather slow (see e.g., Kwok, et al., 1990. Nucl. Acids Res. 18: 999-1005). However, the substitution of an α-thio nucleotide analogs for the subsequent, correct natural deoxynucleotide after the 3′-mismatch termini, the rate of polymerization could be decreased by 5-fold to 13-fold, thus causing a delay in the incorporation of correct nucleotides by the DNA polymerase after the primer comprising a mismatch at the 3′-termini. However, after incorporation of a few bases the rate of DNA synthesis is comparable with the rate observed for. a normal template/primer. Single-base detection by this technique has been improved by incorporation of apyrase to the system, which functions to degrade the nucleotide to a concentration far below the Km of the DNA polymerase. The use of apyrase minimizes further extension upon contact with a mismatched base, and thereby simplifies the data analysis. The above-described technique provides a rapid and real-time analysis for applications in the areas of mutation detection and single-nucleotide polymorphism (SNP) analysis.
  • The pyrophosphate sequencing system takes advantage of the cooperativity of several enzymes to monitor DNA synthesis. Parameters such as stability, fidelity, specificity, sensitivity, K[0132] M and KCAT are of paramount importance for the optimal performance of the enzymes used in the system. In the pyrophosphate sequencing system, the activity of the detection enzymes (i.e., sulfurylase and luciferase) generally remain constant during the sequencing reaction, and are only very slightly inhibited by high amounts of products (see e.g., Ronaghi, et al., 1998. Doctoral Dissertation, The Royal Institute of Technology, Dept. of Biochemistry (Stockholm, Sweden)). Sulfurylase converts PPi to ATP in approximately 2.0 seconds, and the generation of light by luciferase takes place in less than 0.2 seconds. The most critical reactions are the DNA polymerization and the degradation of nucleotides. The value of the enzymes utilized in the pyrophosphate sequencing methodology are listed below:
    Enzyme KM (μM) KCAT (S−1)
    Klenow 0.18 (dTTP) 0.92
    T7 DNA Polymerase 0.36 (dTTP) 0.52
    ATP Sulfurylase 0.56 (APS); 7.0 (PPi) 38
    Firefly Luciferase 20 (ATP) 0.015
    Apyrase 120 (ATP); 260 (ADP) 500 (ATP)
  • The enzymes involved in these two reactions are obviously competing for the same substrate. Therefore, changes in the kinetics of these enzymes directly influence the performance of the sequencing reaction. At the time of dNTP addition, a nucleotide attaches to a polymerase bound to DNA, and polymerization begins. To obtain a rapid polymerization the nucleotide triphosphate concentration must be above the K[0133] M of the DNA polymerase. Conversely, if the concentration of the nucleotide triphosphates is too high, lower fidelity of the polymerase is frequently observed (see e.g., Cline, et al., 1996. PCR fidelity of Pfu DNA polymerase and other thermostable DNA polymerases. Nucl. Acids Res. 24: 3546-3551), although, the KM for the misincorporation rate is much higher than that of the rate for correct incorporation (see e.g., Capson, et al., 1992. Kinetic characterization of the polymerase and exonuclease activity of the gene 43 protein of bacteriophage T4. Biochemistry 31: 10984-10994). Although a very high fidelity can be achieved by using polymerases with inherent exonuclease activity, their use also holds the disadvantage that primer degradation may occur.
  • Although the exonuclease activity of the Klenow fragment of DNA polymerase I (Klenow) is low, it has been demonstrated that the 3′-terminus of the primer was degraded with longer incubations in the absence of nucleotide triphosphates (see e.g., Ronaghi, et al., 1998. [0134] Doctoral Dissertation, The Royal Institute of Technology, Dept. of Biochemistry (Stockholm, Sweden)). Even in the absence of exonuclease activity, an induced-fit binding mechanism in the polymerization step provides a very efficient selectivity for the correct dNTP with a net contribution, approaching a fidelity of 1×105 to 1×106 (see e.g., Wong, et al., 1991. An induced-fit kinetic mechanism for DNA replication fidelity. Biochemistry 30: 526-537). In pyrophosphate sequencing, exonuclease-deficient (exo-) polymerases, such as exo-Klenow or Sequenase®, catalyze the incorporation of a nucleotide only in the presence of a complementary dNTP, confirming a high fidelity of these enzymes even in the absence of proof-reading exonuclease activity. For most polymerases, the KM and KCAT for a the incorporation of a single nucleotide is lower than that of the incorporation of several (see e.g., Van Draanen, et al., 1992. Beta-L-thymidine 5′-triphosphate analogs as DNA polymerase substrates. J. Biol. Chem. 267: 25019-25024). However, the KM values for nucleotides are much lower for DNA polymerases, than for apyrase. An increased fidelity in the system can thus be obtained because the nucleotide concentration necessary for efficient polymerization is relatively low and apyrase degrades nucleotides to a concentration far below the KM of the polymerase in less than 10-15 seconds. The nucleotide-degrading enzyme must possess the following properties: firstly, the enzyme must hydrolyze all deoxynucleotide triphosphates at approximately the same rate. Secondly, it should also hydrolyze ATP to prevent the accumulation of ATP between cycles. Thirdly, the time for nucleotide degradation by the nucleotide-degrading enzyme must be lower than nucleotide incorporation by the polymerase. It is also important that the yield of primer-directed incorporation is as close to 100% as possible before the nucleotide-degrading enzyme has degraded the nucleotide to a concentration below the KM of the polymerase. Changes in other parameters, such as pH, temperature, and ionic concentration may also alter the kinetics of the different enzymes in the system. However, the enzymes typically utilized in the pyrophosphate sequencing system show high stability within a rather broad range of these parameters for several hours (see e.g., Ronaghi, et al., 1998. Doctoral Dissertation, The Royal Institute of Technology, Dept. of Biochemistry (Stockholm, Sweden)).
  • Due to the fact that methodologies currently exist which allow the spectroscopic-detection of single molecules, traditional, the cloning of nucleic acid samples is no longer an absolute requirement in order to obtain nucleic acid sequence information. Currently, a single copy of template which is amplified (e.g., rolling circle amplification) provides a sufficient sample size for the nucleic acid sequencing methodology of the present invention. In brief, the apparatus and methods of the present invention allow the “capture” and quantitation of signals (i.e., photons) within a given optical plane and their subsequent conversion into digital information. Photons are collected from a thin plane roughly equivalent to the volume within which the enzyme and newly synthesized base reside. [0135]
  • Estimates for the spatial and temporal constraints on the pyrophosphate sequencing methodology of the present invention have been calculated, wherein the instant system possesses a 1 cm[0136] 2 area with height approximately 50 μm, for a total volume of 5 μl. With respect to temporal constraints, the molecular species participating in the cascade of reactions are initially defined, wherein:
    N = the DNA attached to the surface
    PP1 =the pyrophosphate molecule released
    ATP = the ATP generated from the pyrophosphate
    L = the light released by luciferase
  • It is further specified that N(0) is the DNA with no nucleotides added, N(1) has 1 nucleotide added, N(2) has 2 nucleotides added, and so on. The pseudo-first-order rate constants which relate the concentrations of molecular species are: [0137]
    N(n) → N(n + 1) + PP1 kN
    PP1 → ATP kP
    ATP → L kA
  • In addition, the diffusion constants D[0138] p for PPi and DA for ATP must also be specified. These values may be estimated from the following exemplar diffusion constants for biomolecules in a dilute water solution (see Weisiger, 1997. Impact of Extracellular and Intracellular Diffusion on Hepatic Uptake Kinetics Department of Medicine and the Liver Center, University of California, San Francisco, Calif., USA, dickw@itsa.ucsf.edu, http://dickw.ucsf.edu/Dapers/goresky97/chapter.html).
    Molecule D/10−5 cm2/sec Method Original Reference
    Albumin 0.066 lag time 1
    Albumin 0.088 light scattering 2
    Water 1.940 NMR 3
  • wherein, Original Reference 1 is: Longsworth, 1954. Temperature dependence of diffusion in aqueous solutions, [0139] J. Phys. Chem. 58: 770-773; Original Reference 2 is: Gaigalas, et al., 1992. Diffusion of bovine serum albumin in aqueous solutions, J. Phys. Chem. 96: 2355-2359; and Original Reference 3 is: Cheng, 1993. Quantitation of non-Einstein diffusion behavior of water in biological tissues by proton NMR diffusion imaging: Synthetic image calculations, Magnet. Reson. Imaging 11: 569-583.
  • In order to estimate the diffusion constant of PP[0140] i, the following exemplar values may be utilized (see CRC Handbook of Chemistry and Physics, 1983. (W. E. Weast. Ed.) CRC Press, Inc., Boca Raton, Fla.):
    Molecule D/10−5 cm2/sec Molecular Weight/amu
    sucrose  0.5226 342.30
    mannitol 0.682 182.18
    penta-erythritol 0.761 136.15
    glycolamide 1.142 N/A
    glycine 1.064  75.07
  • The molecular weight of PP[0141] i is 174 amu. Based upon the aforementioned exemplar values, a diffusion constant of approximately 0.7×10−5 cm2/sec for PPi is expected.
  • It should also be noted that the enzymes catalyzing the three pyrophosphate sequencing reactions are thought to follow Michaelis-Menten kinetics (see e.g. Stryer, 1988. [0142] Biochemistry, W. H. Freeman and Company, New York), which may be described:
  • K M =[E][S]/[ES],
  • velocity=V max [S]/(K M +[S]),
  • V max =k turnover [E T]
  • where [S] is the concentration of substrate, [E] is the concentration of free enzyme, [ES] is the concentration of the enzyme-substrate complex, and [E[0143] T] is the total concentration of enzyme =[E]+[ES].
  • It is preferable that the reaction times are at least as fast as the solution-phase pyrophosphate-based sequencing described in the literature. That rate that a substrate is converted into product is[0144]
  • −d[S]/dt=K turnover [E T ][S]/(K M +[S])
  • The effective concentration of substrate may be estimated from the size of a replicated DNA molecule, at most (10 μm)[0145] 3 and the number of copies (approximately 10,000), yielding a concentration of approximately 17 nm. This is this is smaller than the KM for the enzymes described previously, and therefore the rate can be estimated to be
  • −d[S]/dt=(K turnover /K M)[E T ][S].
  • Thus, with pseudo first-order kinetics, the rate constant for disappearance of substrate depends on K[0146] turnover and KM, which are constants for a given enzyme, and [ET]. Using the same enzyme concentrations reported in the literature will therefore produce similar rates.
  • The first step in the pyrophosphate sequencing reaction (i.e., incorporation of a new nucleotide and release of PP[0147] i) will now be examined in detail. The preferred reaction conditions are: 1 pmol DNA, 3 pmol polymerase, 40 pmol dNTP in 0.2 ml buffer. Under the aforementioned, preferred reaction conditions, the KM for nucleotide incorporation for the Klenow fragment of DNA polymerase I is 0.2 μM and for Sequenase 2.0™ (Promega Biotech, Madison, Wis.) is 0.4 μM, and complete incorporation of 1 base is less than 0.2 sec (see e.g., Ronaghi, et al., 1996. Real-Time DNA Sequencing Using Detection of Pyrophosphate Release, Anal. Biochem. 242: 84-89) with a polymerase concentration of 1.5 nM.
  • In the preferred 5 μl reaction volume, there are a total of 10,000 anchor primers with 10,000 sequencing primer sites each, or 1×10[0148] 8 total extension sites=0.17 fmol. Results which have been previously published in the literature suggest that polymerase should be present at 3-times abundance, or a 0.5 fmol, within the reaction mixture. The final concentration of polymerase is then 0.1 nM. It should be noted that these reaction conditions are readily obtained in the practice of the present invention.
  • As previously stated, the time required for the nucleotide addition reaction is no greater than 0.2 sec per nucleotide. Hence, if the reaction is allowed to proceed for a total of T seconds, then nucleotide addition should be sufficiently rapid that stretches of up to (T/0.2) identical nucleotides should be completely filled-in by the action of the polymerase. As will be discussed infra, the rate-limiting step of the pyrophosphate sequencing reaction is the sulfurylase reaction, which requires a total of approximately 2 sec to complete. Accordingly, a total reaction time which allows completion of the sulfurylase reaction, should be sufficient to allow the polymerase to “fill-in” stretches of up to 10 identical nucleotides. In random DNA species, regions of 10 or more identical nucleotides have been demonstrated to occur with a per-nucleotide probability of approximately 4[0149] −10, which is approximately 1×10−6. In the 10,000 sequences which are extended from anchor primers in a preferred embodiment of the present invention, each of which will be extended at least 30 nt. and preferably 100 nt., it is expected that approximately one run of 10 identical nucleotides will be present. Thus, it may be concluded that runs of identical nucleotides should not pose a difficulty in the practice of the present invention.
  • The overall size of the resulting DNA molecule is, preferably, smaller than the size of the anchoring pads (i.e., 10 μm) and must be smaller than the distance between the individual anchoring pads (i. e., 100 μm). The radius of gyration of a single-stranded DNA concatemer with N total nucleotides may be mathematically-estimated by the following equation: radius=b (N/N[0150] 0)0.6, where b is the persistence length and N0 is the number of nucleotides per persistence length; the exponent 0.6 is characteristic of a self-avoiding walk (see e.g., Doi, 1986. The Theory of polymer Dynamics (Clarendon Press, New York); Flory, 1953. Principles of Polymer Chemistry (Cornell University Press, New York)). Using single-stranded DNA as an example, b is 4 nm and N0 is 13.6 nt. (see e.g., Grosberg, 1994. Statistical Physics of Macromolecules (AIP Press, New York)). Using 10,000 copies of a 100-mer, N=1×106 and the radius of gyration is 3.3 μm.
  • The sulfurylase reaction will now be discussed in detail. The time for the production of ATP from [0151] adenosine 5′-phosphosulfate (APS) and PPi has been estimated to be less than 2 sec (see e.g., Nyrén and Lundin, 1985. Anal. Biochem. 151: 504-509. The reported reaction conditions for 1 pmol PPi in 0.2 ml buffer (5 nM) are 0.3 U/ml ATP sulfurylase (ATP:sulfate adenylyltransferase; Prod. No. A8957; Sigma Chemical Co., St. Louis, Mo.) and 5 μM APS (see e.g., Ronaghi, et al., 1996. Real-Time DNA Sequencing Using Detection of Pyrophosphate Release, Anal. Biochem. 242: 84-89). The manufacturer's information (Sigma Chemical Co., St. Louis, Mo.) for sulfurylase (470 kD) reports an activity of 5-20 units per mg protein (i.e., one unit will produce 1.0 μmole of ATP from APS and PPi per minute at pH 8.0 at 30 C.), whereas the specific activity has been reported elsewhere as 140 units per mg (see Karamohamed, et al., 1999. Purification, and Luminometric Analysis of Recombinant Saccharomyces cerevisiae MET3 Adenosine Triphosphate Sulfurylase Expressed in Escherichia coli, Prot. Express. Purification 15: 381-388). Due to the fact that the reaction conditions utilized in the practice of the present invention are similar to those reaction conditions reported in the aforementioned reference, the sulfurylase concentration within the assay was estimated as 4.6 nM. Thus, at the half-maximal rate, [APS]=0.5 μM and [PPi]=7 μM.
  • In the reaction conditions utilized in the present invention, [PP[0152] i] is approximately 0.17 fmol in 5 μl, or 0.03 nM. The fraction of PPi which is bound to the enzyme is [E]/KM, where [E] is the concentration of free enzyme. Since the enzyme concentration is much larger than the PPi concentration, the total enzyme concentration alone, may be used in the calculations. The fraction of PPi bound to enzyme is found to be 4.6 nM/7 μM=7×10−4. Therefore, it may be concluded that the PPi spends most of its time freely diffusing before being converted to ATP.
  • The mean time for each phosphate (P) to react is 1/kp=2 seconds. The root mean square (RMS) distance it diffuses in each direction is approximately 2D[0153] p/kp, or 2.8×103 μm2. The RMS distance in each direction is 53 μm. This value indicates that each of the individual anchor primers must be more than 50 μm apart, or PPi which is released from one anchor could diffuse to the next, and be detected.
  • Another method which may be used to explain the aforementioned phenomenon is to estimate the amount of PP[0154] i over a first anchor pad that was generated at said first anchor pad relative to the amount of PPi that was generated at a second anchor pad and subsequently diffused over to the location of said first anchor pad. When these two quantities approach each other in magnitude, it become difficult to distinguish the “true” signal from that of the background. This may be mathematically-described by defining a as the radius of an anchor pad and 1/b2 as the density of an anchor pad. Based upon previously published data, a is approximately equal to 10 μm and b is approximately equal to 100 μm. The amount of PPi which is present over said first anchor pad may be described by: exp(−kpt)[1−exp(−a2/2Dpt)] and the amount of PPi present over the second anchor pads may be mathematically-approximated by: (⅓)exp(−kpt)[pa2/b2]exp(−b2/2Dpt). The prefactor ⅓ assumes that ¼ of the DNA sequences will incorporate 1 nucleotide, ¼ of these will then incorporate a second nucleotide, etc., and thus the sum of the series is ⅓. The amounts of PPi over the first and second anchor pads become similar in magnitude when 2Dpt is approximately equal to b2, thus indicating that the RMS distance a molecule diffuses is equal to the distance between adjacent anchor pads. In accord, based upon the assay conditions utilized in the practice of the present invention, the anchor pads must be placed no closer than approximately 50 μm apart, and preferable are at least 3-times further apart (i.e., 150 μm).
  • Although the aforementioned findings set a limit on the surface density of anchor pads, it is possible to decrease the distance requirements, while concomitantly increasing the overall surface density of the anchor pads, by the use of a number of different approaches. One approach is to detect only the early light, although this has the disadvantage of losing signal, particularly from DNA sequences which possess a number of contiguous, identical nucleotides. [0155]
  • A second approach to decrease the distance between anchor pads is to increase the concentration of sulfurylase in the reaction mixture. The reaction rate k[0156] p is directly proportional to the sulfurylase concentration, and the diffusion distance scales as kp −½. Therefore, if the sulfurylase enzyme concentration is increased by a factor of 4-times, the distance between individual anchor pads may be concomitantly reduced by a factor of 2-times.
  • A third approach is to increase the effective concentration of sulfurylase (which will also work for other enzymes described herein) by binding the enzyme to the surface of the anchor pads. The anchor pad can be approximated as one wall of a cubic surface enclosing a sequencing reaction center. Assuming a 10 μm×10 μm surface for the pad, the number of molecules bound to the pad to produce a concentration of a 1 μM is approximately 600,000 molecules. [0157]
  • The sulfurylase concentration in the assay is estimated as 5 nM. The number bound molecules to reach this effective concentration is about 3000 molecules. Thus, by binding more enzyme molecules, a greater effective concentration will be attained. For example, 10,000 molecules could be bound per anchor pad. [0158]
  • As previously estimated, each sulfurylase molecule occupies a total area of 65 nm[0159] 2 on a surface. Accordingly, anchoring a total of 10,000 sulfurylase enzyme molecules on a surface (i.e., so as to equal the 10,000 PPi released) would require 1.7 μm2. This value is only approximately 2% of the available surface area on a 10 μm×10 μm anchor pad. Hence, the concentration of the enzyme may be readily increased to a much higher value.
  • A fourth approach to allow a decrease in the distance between individual anchor pads, is to utilize one or more agents to increase the viscosity of the aqueous-based, pyrophosphate sequencing reagents (e.g., glycerol, polyethylene glycol (PEG), and the like) so as to markedly increase the time it takes for the PPi to diffuse. However, these agents will also concomitantly increase the diffusion time for other non-immobilized components within the sequencing reaction, thus slowing the overall reaction kinetics. Additionally, the use of these agents may also function to chemically-interfere with the sequencing reaction itself. [0160]
  • A fifth, and preferred, methodology to allow a decrease in the distance between individual anchor pads, is to conduct the pyrophosphate sequencing reaction in a spatial-geometry which physically-prevents the released PP[0161] i from diffusing laterally. For example, uniform cavities, which are generated by acid-etching the termini of optical fiber bundles, may be utilized to prevent such lateral diffusion of PPi (see Michael, et al., 1998. Randomly Ordered Addressable High-Density Optical Sensor Arrays, Anal. Chem. 70: 1242-1248). In this embodiment, the important variable involves the total diffusion time for the PPi to exit a cavity of height h, wherein h is the depth of the etched cavity. This diffusion time may be calculated utilizing the equation: 2Dpt=h2. By use of the preferred pyrophosphate sequencing reaction conditions of the present invention in the aforementioned calculations, it may be demonstrated that a cavity 50 μm in depth would be required for the sequencing reaction to proceed to completion before complete diffusion of the PPi from said cavity. Moreover, this type of geometry has the additional advantage of concomitantly reducing background signal from the PPi released from adjacent anchor pads. In contrast to use of a “chip”-based geometry, wherein the required sequencing reagents are “flowed” over the surface of the solid support matrix (i.e., the anchor pads), delivery of the various sequencing reagents in acid-etched optical fiber bundle embodiment is performed by immersion of the acid-etched cavities, alternately, into dNTP/APS/sulfurylase reagents and then, subsequently, into the apyrase reagents to facilitate the degradation of any remaining dNTPs.
  • Subsequently, once ATP has been formed by use of the preferred reaction conditions of the present invention, the reaction time, 1/k[0162] A, has been shown to be 0.2 seconds. Because this reaction time is much lower than the time which the PPi is free to diffuse, it does not significantly alter any of the aforementioned conclusions regarding the assay geometry and conditions utilized in the present invention.
  • In order to mitigate the generation of background light, it is preferable to “localize” (i.e., anchoring or binding) the luciferase in the region of the DNA sequencing templates. It is most preferable to localize the luciferase to a region that is delineated by the distance a PP[0163] i molecule can diffuse before it forms ATP. Methods for binding luciferase to a solid support matrix are well-known in the literature (see e.g., Wang, et al., 1997. Specific Immobilization of Firefly Luciferase through a Biotin Carboxyl Carrier Protein Domain, Analytical Biochem. 246: 133-139). Thus, for a 2 second diffusion time, the luciferase is anchored within a 50 μm distance of the DNA strand. It should be noted, however, that it would be preferable to decrease the diffusion time and thus to further limit the surface area which is required for luciferase binding.
  • In order to determine the concentration of luciferase which it is necessary to bind, previously published conditions were utilized in which luciferase is used at a concentration which gives a response of 200 mV for 0.1 μm ATP (see Ronaghi, et al., 1996. Real-Time DNA Sequencing Using Detection of Pyrophosphate Release, [0164] Analytical Biochem. 242: 84-89). More specifically, it is known from the literature that, in a 0.2 ml reaction volume, 2 ng of luciferase gives a response of 10 mV for 0.1 μM ATP (see Karamohamed and Nyrén, 1999. Real-Time Detection and Quantification of Adenosine Triphosphate Sulfurylase Activity by a Bioluminometric Approach, Analytical Biochem. 271: 81-85). Accordingly, a concentration of 20 ng of luciferase within a 0.2 ml total reaction volume would be required to reproduce these previously-published literature conditions. In the volume of a 10 μm cube around each of the individual anchor pads of the present invention, a luciferase concentration of 1×10−16 grams would be required, and based upon the 71 kD molecular weight of luciferase, this concentration would be equivalent to approximately 1000 luciferase molecules. As previously stated, the surface area of luciferase has been computed at 50 nm2. Thus, assuming the luciferase molecules were biotinylated and bound to the anchor pad, 1000 molecules would occupy a total area of 0.05 μm2. From these calculations it becomes readily apparent that a plethora of luciferase molecules may be bound to the anchor pad, as the area of each anchor pad area is 100 μm2.
  • Again, based upon previously-published results in the literature, each nucleotide takes approximately 3 seconds in toto, to sequence (i.e., 0.5 seconds to add a nucleotide; 2 seconds to make ATP; 0.2 seconds to get fluorescence). Accordingly, a cycle time of approximately 60 seconds per nucleotide is reasonable, requiring approximately 30 minutes per experiment to generate 30 nucleotides of information per sequencing template. [0165]
  • In an alternative embodiment to the aforementioned sequencing methodology (i.e., polymerase→PP[0166] i,→sulfurylase→ATP→luciferase→-light cascade), a polymerase may be developed (e.g., through the use of protein fusion and the like) which possesses the ability to generate light when it incorporates a nucleotide into a growing DNA chain. In yet another alternative embodiment, a sensor may be developed which directly measures the production of PPi in the sequencing reaction. As the production of PPi changes the electric potential of the surrounding buffer, this change could be measured and calibrated to quantitate the concentration of PPi produced.
  • As previously discussed, the polymerase-mediated incorporation of dNTPs into the nucleotide sequence in the pyrophosphate sequencing reaction causes the release of an inorganic pyrophosphate (PPi) moiety which, in turn, through catalysis by luciferase, causes the release of a photon (i.e., light). The photons generated by the pyrophosphate sequencing reaction may subsequently be “captured” and quantitated by a variety of methodologies including, but not limited to: a photomultiplier tube, charge-coupled display (CCD), absorbance photometer, a luminometer, and the like. [0167]
  • The photons generated by the pyrophosphate sequencing reaction are captured by the CCD only if they pass through a focusing device (e.g., an optical lens or optical fiber) and are focused upon a CCD element. The fraction of these photons which are captured may be estimated by the following calculations. First, it is assumed that the lens that focuses the emitted photons is at a distance r from the surface of the solid surface (i.e., DNA chip or etched fiber optic well), where r=1 cm, and that the photons must pass through a region of diameter b (area=πb[0168] 2/4) so as to be focused upon the array element, where b=100 μm. It should also be noted that the emitted photons should escape equally in all directions. At distance r, the photons are dispersed over an area of which is equal to 4πr2. Thus, the fraction of photons which pass through the lens is described by: (½)[1−(1+b2/4r2)−½]. When the value of r is much larger than that of b, the fraction which pass through the lens may then be described by: b2/16r2. For the aforementioned values of r and b, this fraction of photons is 6×10−6.
  • For each nucleotide addition, it is expected that approximately 10,000 PP[0169] i molecules will be generated and, if all are converted by sulfurylase and luciferase, these PPi will result in the emission of approximately 1×104 photons. In order to maximize their subsequent “capture” and quantitation when utilizing a planar array (e.g., a DNA chip), it is preferable to collect the photons immediately at the planar solid support (e.g., the cover slip). This may be accomplished by either: (i) utilizing optical immersion oil between the cover slip and a traditional optical lens or optical fiber bundle or, preferably, (ii) incorporating optical fibers directly into the cover slip itself. Performing the previously described calculations (where in this case, b=100 μm and r=50 μm), the fraction collected is found to be 0.15, which equates to the capture of approximately 1×103 photons. This value would be sufficient to provide an adequate signal.
  • The sequence acquisition software acquires and analyzes the data during the pyrophosphate sequencing cycle. Prior to beginning a given sequencing experiment, a bin of pixels containing each individual reaction center is determined. During each sequencing cycle, four “images” of the entire array are produced, and each image corresponds to excitation of one of the four, fluorescently-labeled nucleotide bases A, C, G, or T (or U). For each reaction center bin, all of the four images are analyzed to determine which nucleotide species has been incorporated at that reaction center during that cycle. As described above, the reaction center bin corresponding to a certain reaction center contains a 10×10 array of pixels. The total number of photons produced by the single fluorophore in that reaction center is determined by the summation of each pixel value in the array. The sums of the reaction center bins from each of the four images are compared, and the image that produces a significant sum corresponds to the newly incorporated base at that reaction center. The images are processed for each of the reaction centers and an array of incorporated nucleotides is recorded. Such processing is capable of being rapidly performed in real-time with modem image processing computers. [0170]
  • Multiple “reads” of individual reaction center arrays may be necessary during the detection step to ensure that the four nucleotides are properly distinguished. Exposure times can be as rapid as 100 msec, with the readout time of the CCD chip being on the order of 250 msec. Thus, the maximum time needed for four complete reads of the array is 1.5 seconds. The total time for a given cycle, including reagent addition, removal, and washes, is certainly less than 10 seconds. Accordingly, a sequencing apparatus consisting of an array of 10,000 reaction centers (i.e., a 100×100 array) is able to detect at least 360 bases per site per hour, or 3.6 Megabases per hour of total sequence, as a conservative estimate. This rate is significantly faster than those of traditional sequencing methodologies. [0171]
  • In addition to short sequencing times, the methods of the present invention do not require the time-consuming processes of initial sample amplification (e.g., cloning or PCR), and gel electrophoresis. The lack of consumables necessary for sample amplification and electrophoresis, coupled with small reagent volumes and reduced manual labor requirements drastically reduce the cost per nucleotide sequenced relative to traditional sequencing techniques. [0172]
  • In one embodiment of the present invention, a nucleic acid sample is sheared prior to inclusion in a reaction center. Once these fragments have been sequenced, sequence analysis software is used to assemble their sequences into contiguous stretches. Many algorithms exist in the art that can compare sequences and deduce their correct overlap. New algorithms have recently been designed to process large amounts of sequence data from shotgun (random) sequencing approaches. [0173]
  • In one preferred embodiment, an algorithm initially reduces the amount of data to be processed by using only two smaller sequences derived from either end of the sequence deduced from a single reaction center in a given experiment. This approach has been proposed for use in shotgun sequencing of the human genome (see e.g., Rawlinson, et al., 1996 [0174] J. Virol 70: 8833-8849; Venter, et al., 1998. Science 280: 1540-1542). It employs algorithms developed at the Institute for Genome Research (TIGR; see e.g., Sutton, et al., 1995. Genome Sci. Technol. 1: 9-16).
  • In an alternative, preferred embodiment, raw data is compressed into a fingerprint of smaller words (e.g., hexanucleotide restriction enzyme sites) and these fingerprints can be compared and assembled into larger continuous blocks of sequence (i.e., contigs). This technique is similar to that used to deduce overlapping sequences after oligonucleotide hybridization (see e.g., Idury and Waterman, 1995. [0175] J. Comput. Biol. 2: 291-306). Yet another embodiment uses existing sequence data, from genetic or physical linkage maps, to assist the assembly of new sequence data from whole genomes or large genomic pieces.
  • The following examples are meant to illustrate, not limit, the invention. [0176]
    • EXAMPLE 1 Construction of Anchor Primers Linked to a Cavitated Terminus Fiber Optic Array
  • The termini of a thin wafer fiber optic array are cavitated by inserting the termini into acid as described by Healey et al., [0177] Anal. Chem. 69: 2213-2216 (1997).
  • A thin layer of a photoactivatable biotin analog is dried onto the cavitated surface as described Hengsakul and Cass ([0178] Biocongiugate Chem. 7: 249-254, 1996) and exposed to white light through a mask to create defined pads, or areas of active biotin. Next, avidin is added and allowed to bind to the biotin. Biotinylated oligonucleotides are then added. The avidin has free biotin binding sites that can anchor biotinylated oligonucleotides through a biotin-avidin-biotin link.
  • The pads are approximately 10 μm on a side with a 100 μm spacing. Oligonucleotides are added so that approximately 37% of the pads include one anchored primer. On a 1 cm surface are deposited 10,000 pads, yielding approximately 3700 pads with a single anchor primer. Sulfurylase, apyrase, and luciferase are also attached to the cavitated substrate using biotin-avidin. [0179]
    • EXAMPLE 2 Annealing and Amplification of Members of a Circular Nucleic Acid Library
  • A library of open circle library templates is prepared from a population of nucleic acids suspected of containing a single nucleotide polymorphism on a 70 bp Sau3A1-MspI fragment. The templates include adapters that are complementary to the anchor primer, a region complementary to a sequencing primer, and an insert sequence that is to be characterized. The library is generated using Sau3A1 and MspI to digest the genomic DNA. Inserts approximately 65-75nucleotides are selected and ligated to adaptor oligonucleotides [0180] 12 nucleotides in length. The adapter oligonucleotides have have sequences complementary to sequences to an anchor primers linked to a substrate surface as described in Example 1.
  • The library is annealed to the array of anchor primers. A DNA polymerase is added, along with dNTPS, and rolling circle replication is used to extend the anchor primer. The result is a single DNA strand, still anchored to the solid support, that is a concatenation of multiple copies of the circular template. 10,000 or more copies of circular templates in the hundred nucleotide size range. [0181]
    • EXAMPLE 3 Sequence Analysis of Nucleic Acid Linked to the Terminus of a Fiber Optic Substrate
  • The fiber optic array wafer containing amplified nucleic acids as described in Example 2 is placed in a perfusion chamber and attached to a bundle of fiber optic arrays, which are themselves linked to a 16 million pixel CCD cameras. A sequencing primer is delivered into the perfusion chamber and allowed to anneal to the amplified sequences. [0182]
  • The sequencing primer primes DNA synthesis extending into the insert suspected of having a polymorphism, as shown in FIG. 1. The sequencing primer is first extended by delivering into the perfusion chamber, in succession, a wash solution, a DNA polymerase, and one of dTTP, dGTP, dCTP, or APS (a dATP analog). The sulfurylase, luciferase, and apyrase, attached to the termini convert any PPi liberated as part of the sequencing reaction to detectable light. The apyrase present degrades any unreacted dNTP. Light is allowed to collect for 3 seconds by a CCD camera linked to the fiber imaging bundle, after which additional wash solution is added to the perfusion chamber for 10 seconds. The next nucleotide is then added, along with polymerase, thereby repeating the cycle. [0183]
  • During the 10 second wash the collected light image is transferred from the CCD camera to a computer. Light emission is analyzed by the computer and used to determine whether the corresponding dNTP has been incorporated into the extended sequence primer. Addition of dNTPS and pyrophosphate sequencing reagents is repeated until the sequence of the insert region containing the suspected polymorphism is obtained. [0184]
    • Other Embodiments
  • It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. [0185]
  • For example, the sequence of the amplified nucleic acid can be determined using by products of RNA synthesis. In this embodiment, an RNA transcript is generated from a promoter sequence present in the circular nucleic acid template library. Suitable promoter sites and their cognate RNA polymerases include RNA polymerases from [0186] E. coli, the RNA polymerase from the bacteriophage T3, the RNA polymerase from the bacteriophage T7, the RNA polymerase from the bacteriophage SP6, and the RNA polymerases from the viral families of bromoviruses, tobamoviruses, tombusvirus, lentiviruses, hepatitis C-like viruses, and picornaviruses. To determine the sequence of an RNA transcript, a predetermined NTP, i.e., an ATP, CTP, GTP, or UTP, is incubated with the template in the presence of the RNA polymerase. Incorporation of the test NTP into a nascent RNA strand can be determined by assaying for the presence of PPi using the enzymatic detection discussed herein.

Claims (62)

What is claimed is:
1. A method for sequencing a nucleic acid, the method comprising:
providing one or more or more nucleic acid anchor primers linked to a solid support;
providing a plurality of circular nucleic acid templates;
annealing an effective amount of the nucleic acid anchor primer to at least one of the single-stranded circular templates to yield a primed anchor primer-circular template complex;
combining the primed anchor primer-circular template complex with a polymerase to generate multiple copies of the circular nucleic acid template;
annealing an effective amount of a sequencing primer to the circular nucleic acid template to yield a primed sequencing primer-circular nucleic acid template complex;
extending the sequencing primer with a polymerase and a predetermined nucleotide triphosphate to yield a sequencing product and a sequencing reaction byproduct; and
identifying the sequencing reaction byproduct, thereby determining the sequence of the nucleic acid.
2. The method of claim 1, wherein the circular nucleic acid template is single-stranded DNA.
3. The method of claim 1, wherein the circular nucleic acid template is an open circle nucleic acid.
4. The method of claim 1, wherein the circular nucleic acid template is a closed circle nucleic acid.
5. The method of claim 1, wherein the circular nucleic acid template is genomic DNA.
6. The method of claim 1, wherein the circular nucleic acid template is cDNA.
7. The method of claim 1, wherein the circular nucleic acid is 10-200 nucleotides in length.
8. The method of claim 1, wherein the circular nucleic acid is 10-100 nucleotides in length.
9. The method of claim 1, wherein the circular nucleic acid is 10-50 nucleotides in length.
10. The method of claim 1, wherein the multiple copies are generated by a polymerase chain reaction.
11. The method of claim 1, wherein the primed circular template is extended by rolling circle amplification to yield a single-stranded concatamer of the annealed circular nucleic acid template.
12. The method of claim 11, further comprising:
annealing a reverse primer to the single-stranded concatamer to yield a primed concatamer template, and
combining the primed concatamer template with a polymerase enzyme to generate multiple copies of the concatamer template.
13. The method of claim 1, wherein the sequencing byproduct is pyrophosphate.
14. The method of claim 13, wherein the pyrophosphate is detected by contacting the sequencing byproduct with ATP sulfurylase under conditions sufficient to form ATP.
15. The method of claim 14, wherein the ATP is detected with luciferase.
16. The method of claim 13, further comprising apyrase.
17. The method of claim 13, further comprising washing the sequencing product with a wash buffer.
18. The method of claim 17, wherein the wash buffer includes apyrase.
19. The method of claim 1, wherein the anchor primer sequence includes a biotin group.
20. The method of claim 19, wherein the biotin group on the anchor primer is linked to an avidin group on the solid support.
21. The method of claim 1, wherein the anchor primer is conjugated to a biotin-BSA moiety.
22. The method of claim 21, wherein the biotin-BSA moiety on the anchor primer is linked to an avidin-biotin group on the solid support.
23. The method of claim 21, wherein the biotin-BSA moiety on the anchor primer is linked to a BSA group on the solid support in the presence of silane.
24. The method of claim 1, wherein the solid support includes at least one optical fiber.
25. The method of claim 1, wherein the sequencing primer is extended in the presence of a dATP analog.
26. The method of claim 25, wherein the dATP analog is adenosine 5′-phosphosulfate (APS).
27. The method of claim 1, wherein the solid substrate includes two or more anchoring primers separated by approximately 10 μm to approximately 200 μm.
28. The method of claim 27, wherein the solid substrate includes two or more anchoring primers separated by approximately 50 μm to approximately 150 μm.
29. The method of claim 27, wherein the solid substrate includes two or more anchoring primers separated by approximately 100 μm to approximately 150 μm.
30. The method of claim 1, wherein the solid support matrix comprises of a plurality of anchor pads that are covalently linked to the solid support.
31. The method of claim 30, wherein the surface area of each anchor pad is approximately 10 μm2.
32. The method of claim 30, wherein and each pad is separated from one another by a distance ranging from approximately 50 μm to approximately 150 μm.
33. A substrate for analyzing a nucleic acid, the substrate comprising:
a cavitated fiber optic surface; and
a nucleic acid sequence linked to the fiber optic surface.
34. The substrate of claim 33, wherein the substrate comprises a plurality of fiber optic surfaces.
35. The substrate of claim 33, wherein the nucleic acid sequence is an anchor primer.
36. The substrate of claim 33, wherein the fiber optic surface includes two or more anchoring primers separated by approximately 10 μm to approximately 200 μm.
37. The substrate of claim 33, wherein the fiber optic surface includes two or more anchoring primers separated by approximately 100 μm to approximately 150 μm.
38. The substrate of claim 33, wherein the fiber optic surface includes two or more anchoring primers separated by approximately 150 μm.
39. The substrate of claim 33, wherein the fiber optic surface includes two or more anchor pads separated by approximately 100 μm to approximately 150 μm.
40. The substrate of claim 39, wherein the surface area of each pad is approximately 10 μm2.
41. A substrate with a cavitated surface comprising 103 or more groups of oligonucleotides covalently attached to the surface in discrete known regions, the 103 or more groups of oligonucleotides occupying a total area of less than 1 cm2 on said substrate, said groups of oligonucleotides having different nucleotide sequences.
42. The substrate as recited in claim 41 wherein said substrate comprises 104 or more different groups of sequences in discrete known regions.
43. The substrate as recited in claim 1 wherein said substrate comprises 105 or more different groups of oligonucleotides with known sequences in discrete known regions.
44. The substrate as recited in claim 1 wherein the groups of oligonucleotides are attached to the surface by a linker.
45. An array of more than 1,000 different groups of oligonucleotide molecules with known sequences covalently coupled to a surface of a cavitated substrate, said groups of oligonucleotide molecules each in discrete known regions and differing from other groups of oligonucleotide molecules in monomer sequence, each of said discrete known regions being an area of less than about 0.01 cm2 and each discrete known region comprising oligonucleotides of known sequence, said different groups occupying a total area of less than 1 cm2.
46. The array as recited in claim 45 wherein said area is less than 10,000 microns2.
47. The array as recited in claim 46 made by the process of:
exposing a first region of said substrate to light to remove photoremovable group from nucleic acids in said first region, and not exposing a second region of said surface to light;
covalently coupling a first nucleotide to said nucleic acids on said part of said substrate exposed to light, said first nucleotide covalently coupled to said photoremovable group;
exposing a part of said first region of said substrate to light, and not exposing another part of said first region of said substrate to light to remove said photoremovable groups; covalently coupling a second nucleotide to said part of said first region exposed to light; and
repeating said steps of exposing said substrate to light and covalently coupling nucleotides until said more than 500 different groups of nucleotides are formed on said surface.
48. The array as recited in claim 46 comprising more than 10,000 groups of oligonucleotides of known sequences.
49. An apparatus for analyzing a nucleic acid sequence, the apparatus comprising:
a perfusion chamber, wherein the chamber includes a nucleic acid substrate;
a conduit in communication with the perfusion chamber;
an imaging system in communication with the perfusion chamber; and
a data collection system in communication with the imaging system.
50. The apparatus of claim 49, wherein the substrate is a planar substrate.
51. The apparatus of claim 49, wherein the imaging system is a fiber optic system.
52. The apparatus of claim 49, wherein the substrate comprises
a cavitated fiber optic surface in communication with said imaging system; and
a nucleic acid sequence linked to the fiber optic surface.
53. The apparatus of claim 49, wherein the substrate comprises a plurality of fiber optic surfaces, said fiber optic surfaces being in communication with said imaging system.
54. The apparatus of claim 49, wherein the fiber optic surface includes two or more anchoring primers separated by approximately 100 μm to approximately 150 μm.
55. The apparatus of claim 49, wherein the fiber optic surface includes two or more anchoring primers separated by approximately 150 μm.
56. The apparatus of claim 49, wherein the fiber optic surface includes two or more anchor pads separated by approximately 100 μm to approximately 150 μm.
57. The apparatus of claim 49, wherein the surface area of each pad is approximately 5 μm2 to approximately 20 μm2.
58. The apparatus of claim 49, wherein the surface area of each pad is approximately 10 μm2.
59. An apparatus for processing a plurality of analyses, the apparatus comprising:
a flow chamber having disposed therein a substrate comprising a plurality of cavitated surfaces, said cavitated surfaces having disposed thereon nucleic acid molecules;
fluid means for delivering processing reagents from one or more reservoirs to the flow chamber so that the analytes anchored to the plurality of microparticles are exposed to the reagents; and
detection means for detecting a sequence of optical signals from each microparticle of the plurality, each optical signal of the sequence being indicative of an interaction between a processing reagent and the analyte anchored thereto, wherein said detection means is in communication with the cavitated surfaces.
60. The apparatus of claim 59, wherein said detection means further comprises signal tracking means for correlating said optical signals from each of said microparticles in each of said digital images to form for each said microparticle of said plurality a sequence of said optical signals.
61. The apparatus of claim 60, wherein said signal tracking means is a CCD camera.
62. The apparatus of claim 59, wherein said analyte is DNA.
US09/826,141 1999-09-16 2001-04-04 Method of sequencing a nucleic acid Abandoned US20020012933A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US09/826,141 US20020012933A1 (en) 1999-09-16 2001-04-04 Method of sequencing a nucleic acid

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US09/398,833 US6274320B1 (en) 1999-09-16 1999-09-16 Method of sequencing a nucleic acid
US09/826,141 US20020012933A1 (en) 1999-09-16 2001-04-04 Method of sequencing a nucleic acid

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
US09/398,833 Division US6274320B1 (en) 1999-09-16 1999-09-16 Method of sequencing a nucleic acid

Publications (1)

Publication Number Publication Date
US20020012933A1 true US20020012933A1 (en) 2002-01-31

Family

ID=23576966

Family Applications (2)

Application Number Title Priority Date Filing Date
US09/398,833 Expired - Lifetime US6274320B1 (en) 1999-09-16 1999-09-16 Method of sequencing a nucleic acid
US09/826,141 Abandoned US20020012933A1 (en) 1999-09-16 2001-04-04 Method of sequencing a nucleic acid

Family Applications Before (1)

Application Number Title Priority Date Filing Date
US09/398,833 Expired - Lifetime US6274320B1 (en) 1999-09-16 1999-09-16 Method of sequencing a nucleic acid

Country Status (6)

Country Link
US (2) US6274320B1 (en)
EP (1) EP1212467A2 (en)
JP (1) JP4727109B2 (en)
AU (1) AU784708B2 (en)
CA (1) CA2384510C (en)
WO (1) WO2001020039A2 (en)

Cited By (48)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040191816A1 (en) * 1992-08-04 2004-09-30 Replicon, Inc. In vitro amplification of nucleic acid molecules via circular replicons
US20060105373A1 (en) * 2004-11-12 2006-05-18 The Board Of Trustees Of The Leland Stanford Junior University Charge perturbation detection system for DNA and other molecules
US20080305535A1 (en) * 1992-08-04 2008-12-11 Replicon, Inc. In vitro amplification of nucleic acid molecules via circular replicons
US20090029385A1 (en) * 2007-07-26 2009-01-29 Pacific Biosciences Of California, Inc. Molecular redundant sequencing
US20090026082A1 (en) * 2006-12-14 2009-01-29 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes using large scale FET arrays
US20090127589A1 (en) * 2006-12-14 2009-05-21 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes using large scale FET arrays
US20090270273A1 (en) * 2008-04-21 2009-10-29 Complete Genomics, Inc. Array structures for nucleic acid detection
US8217433B1 (en) 2010-06-30 2012-07-10 Life Technologies Corporation One-transistor pixel array
US8263336B2 (en) 2009-05-29 2012-09-11 Life Technologies Corporation Methods and apparatus for measuring analytes
US8349167B2 (en) 2006-12-14 2013-01-08 Life Technologies Corporation Methods and apparatus for detecting molecular interactions using FET arrays
US8470164B2 (en) 2008-06-25 2013-06-25 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US8551704B2 (en) 2007-02-16 2013-10-08 Pacific Biosciences Of California, Inc. Controllable strand scission of mini circle DNA
US8552771B1 (en) 2012-05-29 2013-10-08 Life Technologies Corporation System for reducing noise in a chemical sensor array
US8653567B2 (en) 2010-07-03 2014-02-18 Life Technologies Corporation Chemically sensitive sensor with lightly doped drains
US8673627B2 (en) 2009-05-29 2014-03-18 Life Technologies Corporation Apparatus and methods for performing electrochemical reactions
US8685324B2 (en) 2010-09-24 2014-04-01 Life Technologies Corporation Matched pair transistor circuits
US8747748B2 (en) 2012-01-19 2014-06-10 Life Technologies Corporation Chemical sensor with conductive cup-shaped sensor surface
US8776573B2 (en) 2009-05-29 2014-07-15 Life Technologies Corporation Methods and apparatus for measuring analytes
US8821798B2 (en) 2012-01-19 2014-09-02 Life Technologies Corporation Titanium nitride as sensing layer for microwell structure
US8841217B1 (en) 2013-03-13 2014-09-23 Life Technologies Corporation Chemical sensor with protruded sensor surface
US8858782B2 (en) 2010-06-30 2014-10-14 Life Technologies Corporation Ion-sensing charge-accumulation circuits and methods
US8936763B2 (en) 2008-10-22 2015-01-20 Life Technologies Corporation Integrated sensor arrays for biological and chemical analysis
US8962366B2 (en) 2013-01-28 2015-02-24 Life Technologies Corporation Self-aligned well structures for low-noise chemical sensors
US8963216B2 (en) 2013-03-13 2015-02-24 Life Technologies Corporation Chemical sensor with sidewall spacer sensor surface
US9080968B2 (en) 2013-01-04 2015-07-14 Life Technologies Corporation Methods and systems for point of use removal of sacrificial material
US9109251B2 (en) 2004-06-25 2015-08-18 University Of Hawaii Ultrasensitive biosensors
US9116117B2 (en) 2013-03-15 2015-08-25 Life Technologies Corporation Chemical sensor with sidewall sensor surface
US9128044B2 (en) 2013-03-15 2015-09-08 Life Technologies Corporation Chemical sensors with consistent sensor surface areas
WO2016170179A1 (en) 2015-04-24 2016-10-27 Qiagen Gmbh Method for immobilizing a nucleic acid molecule on solid support
WO2016170182A1 (en) 2015-04-24 2016-10-27 Qiagen Gmbh Method for immobilizing a nucleic acid molecule on a solid support
US9618475B2 (en) 2010-09-15 2017-04-11 Life Technologies Corporation Methods and apparatus for measuring analytes
US9671363B2 (en) 2013-03-15 2017-06-06 Life Technologies Corporation Chemical sensor with consistent sensor surface areas
US9823217B2 (en) 2013-03-15 2017-11-21 Life Technologies Corporation Chemical device with thin conductive element
US9835585B2 (en) 2013-03-15 2017-12-05 Life Technologies Corporation Chemical sensor with protruded sensor surface
US9841398B2 (en) 2013-01-08 2017-12-12 Life Technologies Corporation Methods for manufacturing well structures for low-noise chemical sensors
US9970984B2 (en) 2011-12-01 2018-05-15 Life Technologies Corporation Method and apparatus for identifying defects in a chemical sensor array
WO2018137826A1 (en) 2017-01-26 2018-08-02 Qiagen Gmbh Method for enriching template nucleic acids
US10077472B2 (en) 2014-12-18 2018-09-18 Life Technologies Corporation High data rate integrated circuit with power management
US10100357B2 (en) 2013-05-09 2018-10-16 Life Technologies Corporation Windowed sequencing
US10379079B2 (en) 2014-12-18 2019-08-13 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US10451585B2 (en) 2009-05-29 2019-10-22 Life Technologies Corporation Methods and apparatus for measuring analytes
US10458942B2 (en) 2013-06-10 2019-10-29 Life Technologies Corporation Chemical sensor array having multiple sensors per well
US10605767B2 (en) 2014-12-18 2020-03-31 Life Technologies Corporation High data rate integrated circuit with transmitter configuration
US11231451B2 (en) 2010-06-30 2022-01-25 Life Technologies Corporation Methods and apparatus for testing ISFET arrays
US11307166B2 (en) 2010-07-01 2022-04-19 Life Technologies Corporation Column ADC
US11339430B2 (en) 2007-07-10 2022-05-24 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US11378498B2 (en) 2006-06-14 2022-07-05 Verinata Health, Inc. Diagnosis of fetal abnormalities using polymorphisms including short tandem repeats
US11781187B2 (en) * 2006-06-14 2023-10-10 The General Hospital Corporation Rare cell analysis using sample splitting and DNA tags

Families Citing this family (663)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5854033A (en) * 1995-11-21 1998-12-29 Yale University Rolling circle replication reporter systems
US7622294B2 (en) 1997-03-14 2009-11-24 Trustees Of Tufts College Methods for detecting target analytes and enzymatic reactions
US20030027126A1 (en) 1997-03-14 2003-02-06 Walt David R. Methods for detecting target analytes and enzymatic reactions
US7875440B2 (en) 1998-05-01 2011-01-25 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
US20100130368A1 (en) * 1998-07-30 2010-05-27 Shankar Balasubramanian Method and system for sequencing polynucleotides
US20040106110A1 (en) * 1998-07-30 2004-06-03 Solexa, Ltd. Preparation of polynucleotide arrays
US20040121394A1 (en) * 1998-08-19 2004-06-24 Dawson Elliott P. Method for determining polynucleotide sequence variations
US20030138834A1 (en) * 1998-08-19 2003-07-24 Dawson Elliott P. Method for determining polynucleotide sequence variations
US7079241B2 (en) 2000-04-06 2006-07-18 Invitrogen Corp. Spatial positioning of spectrally labeled beads
US6531302B1 (en) * 1999-04-12 2003-03-11 Nanogen/Becton Dickinson Partnership Anchored strand displacement amplification on an electronically addressable microchip
US6238868B1 (en) * 1999-04-12 2001-05-29 Nanogen/Becton Dickinson Partnership Multiplex amplification and separation of nucleic acid sequences using ligation-dependant strand displacement amplification and bioelectronic chip technology
JP2002541475A (en) * 1999-04-12 2002-12-03 ナノゲン／ベクトン・ディッキンソン・パートナーシップ Amplification and separation of nucleic acid sequences using strand displacement amplification and bioelectronic microchip technology
US6326173B1 (en) * 1999-04-12 2001-12-04 Nanogen/Becton Dickinson Partnership Electronically mediated nucleic acid amplification in NASBA
WO2000063437A2 (en) † 1999-04-20 2000-10-26 Illumina, Inc. Detection of nucleic acid reactions on bead arrays
US20030207295A1 (en) * 1999-04-20 2003-11-06 Kevin Gunderson Detection of nucleic acid reactions on bead arrays
US20030108867A1 (en) * 1999-04-20 2003-06-12 Chee Mark S Nucleic acid sequencing using microsphere arrays
US20050244870A1 (en) * 1999-04-20 2005-11-03 Illumina, Inc. Nucleic acid sequencing using microsphere arrays
US20060275782A1 (en) 1999-04-20 2006-12-07 Illumina, Inc. Detection of nucleic acid reactions on bead arrays
US6818395B1 (en) 1999-06-28 2004-11-16 California Institute Of Technology Methods and apparatus for analyzing polynucleotide sequences
US7244559B2 (en) * 1999-09-16 2007-07-17 454 Life Sciences Corporation Method of sequencing a nucleic acid
US7582420B2 (en) * 2001-07-12 2009-09-01 Illumina, Inc. Multiplex nucleic acid reactions
US6770441B2 (en) 2000-02-10 2004-08-03 Illumina, Inc. Array compositions and methods of making same
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
US9708358B2 (en) 2000-10-06 2017-07-18 The Trustees Of Columbia University In The City Of New York Massive parallel method for decoding DNA and RNA
AU2002241803A1 (en) * 2000-10-20 2002-06-18 The Board Of Trustees Of The Leland Stanford Junior University Transient electrical signal based methods and devices for characterizing molecular interaction and/or motion in a sample
AU2002227156A1 (en) 2000-12-01 2002-06-11 Visigen Biotechnologies, Inc. Enzymatic nucleic acid synthesis: compositions and methods for altering monomer incorporation fidelity
GB0103622D0 (en) * 2001-02-14 2001-03-28 Univ Cambridge Tech Methods for detecting DNA polymerisation
JP2004523243A (en) 2001-03-12 2004-08-05 カリフォルニア インスティチュート オブ テクノロジー Method and apparatus for analyzing polynucleotide sequences by asynchronous base extension
FR2822848B1 (en) * 2001-03-28 2004-05-07 Genesystems INTEGRATED AND MINIATURE NUCLEIC ACID SEQUENCING DEVICE
US20030104421A1 (en) * 2001-05-07 2003-06-05 Colangelo Christopher M. Methods and compositions for nucleic acid amplification
WO2004059289A2 (en) * 2001-05-22 2004-07-15 Epicentre Technologies Target-dependent transcription using deletion mutants of n4 rna polymerase
EP1415004A4 (en) * 2001-07-03 2005-01-19 Univ Leland Stanford Junior BIOLUMINESCENCE REGENERATIVE CYCLE (BRC) FOR THE QUANTIFICATION OF NUCLEIC ACIDS
WO2003004690A2 (en) * 2001-07-06 2003-01-16 454$m(3) CORPORATION Method for isolation of independent, parallel chemical micro-reactions using a porous filter
GB2378245A (en) * 2001-08-03 2003-02-05 Mats Nilsson Nucleic acid amplification method
US6956114B2 (en) 2001-10-30 2005-10-18 '454 Corporation Sulfurylase-luciferase fusion proteins and thermostable sulfurylase
US6902921B2 (en) 2001-10-30 2005-06-07 454 Corporation Sulfurylase-luciferase fusion proteins and thermostable sulfurylase
US20050124022A1 (en) * 2001-10-30 2005-06-09 Maithreyan Srinivasan Novel sulfurylase-luciferase fusion proteins and thermostable sulfurylase
EP1478783A4 (en) * 2002-02-06 2006-12-27 Ge Healthcare Bio Sciences Ab COMPOSITIONS AND METHODS FOR ROLLING CIRCLE AMPLIFICATION
US7553619B2 (en) 2002-02-08 2009-06-30 Qiagen Gmbh Detection method using dissociated rolling circle amplification
AU2003234255A1 (en) * 2002-04-26 2003-11-10 Lynx Therapeutics, Inc. Constant length signatures for parallel sequencing of polynucleotides
US20030215816A1 (en) * 2002-05-20 2003-11-20 Narayan Sundararajan Method for sequencing nucleic acids by observing the uptake of nucleotides modified with bulky groups
US6952651B2 (en) * 2002-06-17 2005-10-04 Intel Corporation Methods and apparatus for nucleic acid sequencing by signal stretching and data integration
US20040197793A1 (en) * 2002-08-30 2004-10-07 Arjang Hassibi Methods and apparatus for biomolecule detection, identification, quantification and/or sequencing
US20040197845A1 (en) * 2002-08-30 2004-10-07 Arjang Hassibi Methods and apparatus for pathogen detection, identification and/or quantification
US7595883B1 (en) 2002-09-16 2009-09-29 The Board Of Trustees Of The Leland Stanford Junior University Biological analysis arrangement and approach therefor
SE0202867D0 (en) * 2002-09-27 2002-09-27 Pyrosequencing Ab New sequencing method
US20040067492A1 (en) * 2002-10-04 2004-04-08 Allan Peng Reverse transcription on microarrays
EP2112229A3 (en) 2002-11-25 2009-12-02 Sequenom, Inc. Methods for identifying risk of breast cancer and treatments thereof
US20040121338A1 (en) * 2002-12-19 2004-06-24 Alsmadi Osama A. Real-time detection of rolling circle amplification products
US9487823B2 (en) 2002-12-20 2016-11-08 Qiagen Gmbh Nucleic acid amplification
US6977153B2 (en) * 2002-12-31 2005-12-20 Qiagen Gmbh Rolling circle amplification of RNA
WO2004065634A1 (en) * 2003-01-15 2004-08-05 Bioventures, Inc. Method for determining polynucleotide sequence variations
CA2513899C (en) 2003-01-29 2013-03-26 454 Corporation Methods of amplifying and sequencing nucleic acids
EP1592810A2 (en) * 2003-02-12 2005-11-09 Genizon Svenska AB Methods and means for nucleic acid sequencing
US6943768B2 (en) 2003-02-21 2005-09-13 Xtellus Inc. Thermal control system for liquid crystal cell
GB0307403D0 (en) 2003-03-31 2003-05-07 Medical Res Council Selection by compartmentalised screening
US8043834B2 (en) 2003-03-31 2011-10-25 Qiagen Gmbh Universal reagents for rolling circle amplification and methods of use
US20060078893A1 (en) 2004-10-12 2006-04-13 Medical Research Council Compartmentalised combinatorial chemistry by microfluidic control
GB0307428D0 (en) 2003-03-31 2003-05-07 Medical Res Council Compartmentalised combinatorial chemistry
US20040248103A1 (en) * 2003-06-04 2004-12-09 Feaver William John Proximity-mediated rolling circle amplification
US7169560B2 (en) 2003-11-12 2007-01-30 Helicos Biosciences Corporation Short cycle methods for sequencing polynucleotides
WO2005054431A2 (en) * 2003-12-01 2005-06-16 454 Corporation Method for isolation of independent, parallel chemical micro-reactions using a porous filter
US7927797B2 (en) 2004-01-28 2011-04-19 454 Life Sciences Corporation Nucleic acid amplification with continuous flow emulsion
WO2005080605A2 (en) 2004-02-19 2005-09-01 Helicos Biosciences Corporation Methods and kits for analyzing polynucleotide sequences
GB2413796B (en) * 2004-03-25 2006-03-29 Global Genomics Ab Methods and means for nucleic acid sequencing
US20050221339A1 (en) 2004-03-31 2005-10-06 Medical Research Council Harvard University Compartmentalised screening by microfluidic control
SE0401270D0 (en) 2004-05-18 2004-05-18 Fredrik Dahl Method for amplifying specific nucleic acids in parallel
US7622281B2 (en) * 2004-05-20 2009-11-24 The Board Of Trustees Of The Leland Stanford Junior University Methods and compositions for clonal amplification of nucleic acid
US7635562B2 (en) 2004-05-25 2009-12-22 Helicos Biosciences Corporation Methods and devices for nucleic acid sequence determination
US7476734B2 (en) 2005-12-06 2009-01-13 Helicos Biosciences Corporation Nucleotide analogs
JP5037343B2 (en) * 2004-06-10 2012-09-26 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 Nucleic acid analysis method
US20060024711A1 (en) * 2004-07-02 2006-02-02 Helicos Biosciences Corporation Methods for nucleic acid amplification and sequence determination
US7566532B1 (en) 2004-07-02 2009-07-28 Quest Diagnostics Investments Incorporated Methods for detecting retroviruses
CA2579150C (en) * 2004-09-17 2014-11-25 Pacific Biosciences Of California, Inc. Apparatus and method for analysis of molecules
US7170050B2 (en) * 2004-09-17 2007-01-30 Pacific Biosciences Of California, Inc. Apparatus and methods for optical analysis of molecules
US7968287B2 (en) 2004-10-08 2011-06-28 Medical Research Council Harvard University In vitro evolution in microfluidic systems
JP2008520245A (en) * 2004-11-23 2008-06-19 ワン，シャオ・ビン Detection of nucleic acid diversity by cleavage-amplification method
US7220549B2 (en) 2004-12-30 2007-05-22 Helicos Biosciences Corporation Stabilizing a nucleic acid for nucleic acid sequencing
US7482120B2 (en) 2005-01-28 2009-01-27 Helicos Biosciences Corporation Methods and compositions for improving fidelity in a nucleic acid synthesis reaction
US8309303B2 (en) 2005-04-01 2012-11-13 Qiagen Gmbh Reverse transcription and amplification of RNA with simultaneous degradation of DNA
US7785862B2 (en) 2005-04-07 2010-08-31 454 Life Sciences Corporation Thin film coated microwell arrays
US7682816B2 (en) 2005-04-07 2010-03-23 454 Life Sciences Corporation Thin film coated microwell arrays and methods of using same
US20060228721A1 (en) 2005-04-12 2006-10-12 Leamon John H Methods for determining sequence variants using ultra-deep sequencing
ES2404311T3 (en) 2005-04-12 2013-05-27 454 Life Sciences Corporation Methods for determining sequence variants using ultra-deep sequencing
EP3257949A1 (en) 2005-06-15 2017-12-20 Complete Genomics Inc. Nucleic acid analysis by random mixtures of non-overlapping fragments
WO2007016269A2 (en) * 2005-07-29 2007-02-08 Applera Corporation Detection of polyphosphate using fluorescently labeled polyphosphate acceptor substrates
KR20080050584A (en) * 2005-08-19 2008-06-09 커먼웰쓰 사이언티픽 앤 인더스트리알 리서치 오거니제이션 Arachnocampa Luciferase
US7666593B2 (en) 2005-08-26 2010-02-23 Helicos Biosciences Corporation Single molecule sequencing of captured nucleic acids
EP1762627A1 (en) 2005-09-09 2007-03-14 Qiagen GmbH Method for the activation of a nucleic acid for performing a polymerase reaction
US7960104B2 (en) * 2005-10-07 2011-06-14 Callida Genomics, Inc. Self-assembled single molecule arrays and uses thereof
JP2009513135A (en) 2005-10-28 2009-04-02 プリーシス・ファーマシューティカルズ・インコーポレイテッド Methods for identifying compounds of interest using encoded libraries
PT3305900T (en) 2005-12-01 2021-10-27 Nuevolution As Enzymatic encoding methods for efficient synthesis of large libraries
WO2007081385A2 (en) 2006-01-11 2007-07-19 Raindance Technologies, Inc. Microfluidic devices and methods of use in the formation and control of nanoreactors
US8364417B2 (en) 2007-02-15 2013-01-29 454 Life Sciences Corporation System and method to correct out of phase errors in DNA sequencing data by use of a recursive algorithm
US8301394B2 (en) * 2006-02-16 2012-10-30 454 Life Sciences Corporation System and method for correcting primer extension errors in nucleic acid sequence data
CN101432439B (en) * 2006-02-24 2013-07-24 考利达基因组股份有限公司 High throughput genome sequencing on DNA arrays
SG170028A1 (en) * 2006-02-24 2011-04-29 Callida Genomics Inc High throughput genome sequencing on dna arrays
US7397546B2 (en) 2006-03-08 2008-07-08 Helicos Biosciences Corporation Systems and methods for reducing detected intensity non-uniformity in a laser beam
US9562837B2 (en) 2006-05-11 2017-02-07 Raindance Technologies, Inc. Systems for handling microfludic droplets
EP2530167A1 (en) 2006-05-11 2012-12-05 Raindance Technologies, Inc. Microfluidic Devices
EP3617321B1 (en) 2006-05-31 2024-10-23 Sequenom, Inc. Kit for the extraction and amplification of nucleic acid from a sample
CA2654165A1 (en) 2006-06-05 2007-12-13 Cancer Care Ontario Assessment of risk for colorectal cancer
EP2077912B1 (en) 2006-08-07 2019-03-27 The President and Fellows of Harvard College Fluorocarbon emulsion stabilizing surfactants
GB0618046D0 (en) * 2006-09-13 2006-10-25 Lingvitae As Method
US7910354B2 (en) 2006-10-27 2011-03-22 Complete Genomics, Inc. Efficient arrays of amplified polynucleotides
US20090111706A1 (en) 2006-11-09 2009-04-30 Complete Genomics, Inc. Selection of dna adaptor orientation by amplification
US20080221832A1 (en) * 2006-11-09 2008-09-11 Complete Genomics, Inc. Methods for computing positional base probabilities using experminentals base value distributions
US20080242560A1 (en) * 2006-11-21 2008-10-02 Gunderson Kevin L Methods for generating amplified nucleic acid arrays
US7989185B2 (en) * 2006-11-29 2011-08-02 The Board Of Trustees Of The Leland Stanford Junior University Rapid, informative diagnostic assay for influenza viruses including H5N1
GB2457402B (en) 2006-12-01 2011-10-19 Univ Columbia Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators
US7902345B2 (en) 2006-12-05 2011-03-08 Sequenom, Inc. Detection and quantification of biomolecules using mass spectrometry
US7932034B2 (en) * 2006-12-20 2011-04-26 The Board Of Trustees Of The Leland Stanford Junior University Heat and pH measurement for sequencing of DNA
JP2008154467A (en) * 2006-12-21 2008-07-10 Olympus Corp Method for amplifying nucleic acid and method for analyzing nucleic acid using the same
WO2008097559A2 (en) 2007-02-06 2008-08-14 Brandeis University Manipulation of fluids and reactions in microfluidic systems
AU2008213634B2 (en) 2007-02-08 2013-09-05 Sequenom, Inc. Nucleic acid-based tests for RhD typing, gender determination and nucleic acid quantification
EP2243834A1 (en) 2007-03-05 2010-10-27 Cancer Care Ontario Assessment of risk for colorectal cancer
US8617816B2 (en) * 2007-03-16 2013-12-31 454 Life Sciences, A Roche Company System and method for detection of HIV drug resistant variants
US8652780B2 (en) 2007-03-26 2014-02-18 Sequenom, Inc. Restriction endonuclease enhanced polymorphic sequence detection
US8592221B2 (en) 2007-04-19 2013-11-26 Brandeis University Manipulation of fluids, fluid components and reactions in microfluidic systems
JP2010528608A (en) * 2007-06-01 2010-08-26 ４５４ ライフ サイエンシーズ コーポレイション System and method for identifying individual samples from complex mixtures
US20090053724A1 (en) * 2007-06-28 2009-02-26 454 Life Sciences Corporation System and method for adaptive reagent control in nucleic acid sequencing
WO2009032781A2 (en) 2007-08-29 2009-03-12 Sequenom, Inc. Methods and compositions for universal size-specific polymerase chain reaction
FR2920598B1 (en) * 2007-09-03 2010-08-27 Eurocopter France ELECTRICAL CONNECTION OF A FIRST AND A SECOND ELECTRICALLY ISOLATED ELECTRICALLY INSULATED METALLIC PIECES
EP2201136B1 (en) 2007-10-01 2017-12-06 Nabsys 2.0 LLC Nanopore sequencing by hybridization of probes to form ternary complexes and variable range alignment
CN101168773B (en) * 2007-10-16 2010-06-02 东南大学 A nucleic acid sequencing method based on fluorescence quenching
US20100086914A1 (en) * 2008-10-03 2010-04-08 Roche Molecular Systems, Inc. High resolution, high throughput hla genotyping by clonal sequencing
WO2009051807A1 (en) 2007-10-19 2009-04-23 The Trustees Of Columbia University In The City Of New York Design and synthesis of cleavable fluorescent nucleotides as reversible terminators for dna sequencing by synthesis
DK2725107T3 (en) 2007-10-19 2019-01-02 Univ Columbia DNA Sequencing with Non-Fluorescent Nucleotide Reversible Terminators and ddNTPs Modified by Split Label and Nucleic Acid comprising Inosine with Reversible Terminators
US8415099B2 (en) 2007-11-05 2013-04-09 Complete Genomics, Inc. Efficient base determination in sequencing reactions
US8518640B2 (en) * 2007-10-29 2013-08-27 Complete Genomics, Inc. Nucleic acid sequencing and process
US7897344B2 (en) * 2007-11-06 2011-03-01 Complete Genomics, Inc. Methods and oligonucleotide designs for insertion of multiple adaptors into library constructs
US8298768B2 (en) * 2007-11-29 2012-10-30 Complete Genomics, Inc. Efficient shotgun sequencing methods
WO2009061840A1 (en) * 2007-11-05 2009-05-14 Complete Genomics, Inc. Methods and oligonucleotide designs for insertion of multiple adaptors employing selective methylation
US8592150B2 (en) 2007-12-05 2013-11-26 Complete Genomics, Inc. Methods and compositions for long fragment read sequencing
WO2009091934A1 (en) 2008-01-17 2009-07-23 Sequenom, Inc. Single molecule nucleic acid sequence analysis processes and compositions
WO2009094583A1 (en) * 2008-01-23 2009-07-30 Complete Genomics, Inc. Methods and compositions for preventing bias in amplification and sequencing reactions
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
US20090203086A1 (en) * 2008-02-06 2009-08-13 454 Life Sciences Corporation System and method for improved signal detection in nucleic acid sequencing
WO2009120808A2 (en) 2008-03-26 2009-10-01 Sequenom, Inc. Restriction endonuclease enhanced polymorphic sequence detection
EP4230747A3 (en) 2008-03-28 2023-11-15 Pacific Biosciences Of California, Inc. Compositions and methods for nucleic acid sequencing
WO2009140662A1 (en) * 2008-05-15 2009-11-19 Illumina, Inc. Engineered luciferases
US7888034B2 (en) 2008-07-01 2011-02-15 454 Life Sciences Corporation System and method for detection of HIV tropism variants
US12038438B2 (en) 2008-07-18 2024-07-16 Bio-Rad Laboratories, Inc. Enzyme quantification
EP4047367A1 (en) 2008-07-18 2022-08-24 Bio-Rad Laboratories, Inc. Method for detecting target analytes with droplet libraries
US8262879B2 (en) 2008-09-03 2012-09-11 Nabsys, Inc. Devices and methods for determining the length of biopolymers and distances between probes bound thereto
US9650668B2 (en) 2008-09-03 2017-05-16 Nabsys 2.0 Llc Use of longitudinally displaced nanoscale electrodes for voltage sensing of biomolecules and other analytes in fluidic channels
CN102186989B (en) 2008-09-03 2021-06-29 纳伯塞斯2.0有限责任公司 Use of longitudinally displaced nanoscale electrodes for voltage sensing of biomolecules and other analytes in fluidic channels
US8383345B2 (en) 2008-09-12 2013-02-26 University Of Washington Sequence tag directed subassembly of short sequencing reads into long sequencing reads
US8476013B2 (en) 2008-09-16 2013-07-02 Sequenom, Inc. Processes and compositions for methylation-based acid enrichment of fetal nucleic acid from a maternal sample useful for non-invasive prenatal diagnoses
US8962247B2 (en) 2008-09-16 2015-02-24 Sequenom, Inc. Processes and compositions for methylation-based enrichment of fetal nucleic acid from a maternal sample useful for non invasive prenatal diagnoses
US20100261189A1 (en) * 2008-10-03 2010-10-14 Roche Molecular Systems, Inc. System and method for detection of HLA Variants
US8486865B2 (en) 2008-11-03 2013-07-16 The Regents Of The University Of California Methods for detecting modification resistant nucleic acids
US8486630B2 (en) 2008-11-07 2013-07-16 Industrial Technology Research Institute Methods for accurate sequence data and modified base position determination
EP2607496B1 (en) 2008-12-23 2014-07-16 Illumina, Inc. Methods useful in nucleic acid sequencing protocols
EP2391732B1 (en) * 2009-01-30 2015-05-27 Oxford Nanopore Technologies Limited Methods using adaptors for nucleic acid constructs in transmembrane sequencing
EP3722810A3 (en) 2009-02-11 2021-01-13 Caris MPI, Inc. Molecular profiling of tumors
DK2401607T3 (en) 2009-02-25 2016-02-29 Illumina Inc Separation of pyrophosphatfrigivelse and pyrophosphatdetektering
WO2010111231A1 (en) 2009-03-23 2010-09-30 Raindance Technologies, Inc. Manipulation of microfluidic droplets
US8455260B2 (en) 2009-03-27 2013-06-04 Massachusetts Institute Of Technology Tagged-fragment map assembly
EP3514244B1 (en) 2009-04-03 2021-07-07 Sequenom, Inc. Nucleic acid preparation methods
US12129514B2 (en) 2009-04-30 2024-10-29 Molecular Loop Biosolutions, Llc Methods and compositions for evaluating genetic markers
EP2425240A4 (en) 2009-04-30 2012-12-12 Good Start Genetics Inc Methods and compositions for evaluating genetic markers
WO2010127304A2 (en) * 2009-05-01 2010-11-04 Illumina, Inc. Sequencing methods
US8246799B2 (en) * 2009-05-28 2012-08-21 Nabsys, Inc. Devices and methods for analyzing biomolecules and probes bound thereto
US8574835B2 (en) 2009-05-29 2013-11-05 Life Technologies Corporation Scaffolded nucleic acid polymer particles and methods of making and using
US9524369B2 (en) 2009-06-15 2016-12-20 Complete Genomics, Inc. Processing and analysis of complex nucleic acid sequence data
ES2661055T3 (en) 2009-09-22 2018-03-27 Roche Diagniostics Gmbh Determination of KIR haplotypes by exon amplification
US10520500B2 (en) 2009-10-09 2019-12-31 Abdeslam El Harrak Labelled silica-based nanomaterial with enhanced properties and uses thereof
US8609339B2 (en) 2009-10-09 2013-12-17 454 Life Sciences Corporation System and method for emulsion breaking and recovery of biological elements
WO2011044437A2 (en) * 2009-10-09 2011-04-14 Stc.Unm Polony sequencing methods
EP2494077A4 (en) 2009-10-27 2013-08-21 Caris Mpi Inc MOLECULAR PROFILING FOR PERSONALIZED MEDICINE
ES2577017T3 (en) 2009-12-22 2016-07-12 Sequenom, Inc. Procedures and kits to identify aneuploidy
US10837883B2 (en) 2009-12-23 2020-11-17 Bio-Rad Laboratories, Inc. Microfluidic systems and methods for reducing the exchange of molecules between droplets
WO2011093939A1 (en) 2010-02-01 2011-08-04 Illumina, Inc. Focusing methods and optical systems and assemblies using the same
US10351905B2 (en) 2010-02-12 2019-07-16 Bio-Rad Laboratories, Inc. Digital analyte analysis
EP4484577A3 (en) 2010-02-12 2025-03-26 Bio-Rad Laboratories, Inc. Digital analyte analysis
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
CN202281746U (en) 2010-03-06 2012-06-20 伊鲁米那股份有限公司 Measuring equipment for detecting optical signal from sample as well as optical module and optical system for measuring equipment
US20190300945A1 (en) 2010-04-05 2019-10-03 Prognosys Biosciences, Inc. Spatially Encoded Biological Assays
WO2011127099A1 (en) 2010-04-05 2011-10-13 Prognosys Biosciences, Inc. Spatially encoded biological assays
US10787701B2 (en) 2010-04-05 2020-09-29 Prognosys Biosciences, Inc. Spatially encoded biological assays
EP3540059A1 (en) 2010-04-16 2019-09-18 Nuevolution A/S Bi-functional complexes and methods for making and using such complexes
CA2793970A1 (en) 2010-04-30 2011-11-03 F. Hoffmann-La Roche Ag System and method for purification and use of inorganic pyrophosphatase from aquifex aeolicus
US20110287432A1 (en) 2010-05-21 2011-11-24 454 Life Science Corporation System and method for tailoring nucleotide concentration to enzymatic efficiencies in dna sequencing technologies
AU2011226792A1 (en) 2010-06-11 2012-01-12 Life Technologies Corporation Alternative nucleotide flows in sequencing-by-synthesis methods
WO2012027224A1 (en) 2010-08-24 2012-03-01 Dana-Farber Cancer Institute, Inc. Methods for predicting anti-cancer response
US9029103B2 (en) 2010-08-27 2015-05-12 Illumina Cambridge Limited Methods for sequencing polynucleotides
US8483969B2 (en) 2010-09-17 2013-07-09 Illuminia, Inc. Variation analysis for multiple templates on a solid support
US8715933B2 (en) 2010-09-27 2014-05-06 Nabsys, Inc. Assay methods using nicking endonucleases
WO2012050920A1 (en) 2010-09-29 2012-04-19 Illumina, Inc. Compositions and methods for sequencing nucleic acids
US20120077716A1 (en) 2010-09-29 2012-03-29 454 Life Sciences Corporation System and method for producing functionally distinct nucleic acid library ends through use of deoxyinosine
US9562897B2 (en) 2010-09-30 2017-02-07 Raindance Technologies, Inc. Sandwich assays in droplets
US20120322665A1 (en) 2010-10-08 2012-12-20 454 Life Sciences Corporation System and method for detection of hiv-1 clades and recombinants of the reverse transcriptase and protease regions
EP2633069B1 (en) 2010-10-26 2015-07-01 Illumina, Inc. Sequencing methods
US8666678B2 (en) 2010-10-27 2014-03-04 Life Technologies Corporation Predictive model for use in sequencing-by-synthesis
US10273540B2 (en) 2010-10-27 2019-04-30 Life Technologies Corporation Methods and apparatuses for estimating parameters in a predictive model for use in sequencing-by-synthesis
US8575071B2 (en) 2010-11-03 2013-11-05 Illumina, Inc. Reducing adapter dimer formation
WO2012061832A1 (en) 2010-11-05 2012-05-10 Illumina, Inc. Linking sequence reads using paired code tags
US9074251B2 (en) 2011-02-10 2015-07-07 Illumina, Inc. Linking sequence reads using paired code tags
US8859201B2 (en) 2010-11-16 2014-10-14 Nabsys, Inc. Methods for sequencing a biomolecule by detecting relative positions of hybridized probes
WO2012074855A2 (en) 2010-11-22 2012-06-07 The Regents Of The University Of California Methods of identifying a cellular nascent rna transcript
EP2652659B1 (en) 2010-12-14 2020-04-15 Life Technologies Corporation Systems and methods for run-time sequencing run quality monitoring
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
EP2659408B1 (en) 2010-12-29 2019-03-27 Life Technologies Corporation Time-warped background signal for sequencing-by-synthesis operations
US20130060482A1 (en) 2010-12-30 2013-03-07 Life Technologies Corporation Methods, systems, and computer readable media for making base calls in nucleic acid sequencing
EP3582224B1 (en) 2010-12-30 2026-04-29 Life Technologies Corporation Models for analyzing data from sequencing-by-synthesis operations
US10241075B2 (en) 2010-12-30 2019-03-26 Life Technologies Corporation Methods, systems, and computer readable media for nucleic acid sequencing
US8951781B2 (en) 2011-01-10 2015-02-10 Illumina, Inc. Systems, methods, and apparatuses to image a sample for biological or chemical analysis
WO2012100194A1 (en) 2011-01-20 2012-07-26 Ibis Biosciences, Inc. Microfluidic transducer
CN103443338B (en) 2011-02-02 2017-09-22 华盛顿大学商业化中心 Massively parallel continguity mapping
EP3412778A1 (en) 2011-02-11 2018-12-12 Raindance Technologies, Inc. Methods for forming mixed droplets
WO2012109574A2 (en) 2011-02-11 2012-08-16 Nabsys, Inc. Assay methods using dna binding proteins
WO2012112804A1 (en) 2011-02-18 2012-08-23 Raindance Technoligies, Inc. Compositions and methods for molecular labeling
US20120244523A1 (en) 2011-03-25 2012-09-27 454 Life Sciences Corporation System and Method for Detection of HIV Integrase Variants
CN103764845B (en) 2011-04-08 2016-02-17 生命科技股份有限公司 For phase-protected reagent flow sequencing in sequencing-by-synthesis
GB201106254D0 (en) 2011-04-13 2011-05-25 Frisen Jonas Method and product
EP3216872B1 (en) 2011-06-02 2020-04-01 Bio-Rad Laboratories, Inc. Enzyme quantification
US8841071B2 (en) 2011-06-02 2014-09-23 Raindance Technologies, Inc. Sample multiplexing
EP2718465B1 (en) 2011-06-09 2022-04-13 Illumina, Inc. Method of making an analyte array
JP6117194B2 (en) 2011-06-17 2017-04-19 ミリアド・ジェネティックス・インコーポレイテッド Methods and materials for assessing allelic imbalance
US8658430B2 (en) 2011-07-20 2014-02-25 Raindance Technologies, Inc. Manipulating droplet size
JP6298404B2 (en) 2011-07-25 2018-03-20 オックスフォード ナノポール テクノロジーズ リミテッド Hairpin loop method for double-stranded polynucleotide sequencing using transmembrane pores
US20150087537A1 (en) 2011-08-31 2015-03-26 Life Technologies Corporation Methods, Systems, Computer Readable Media, and Kits for Sample Identification
US10704164B2 (en) 2011-08-31 2020-07-07 Life Technologies Corporation Methods, systems, computer readable media, and kits for sample identification
EP3620533B1 (en) 2011-09-06 2023-01-18 Gen-Probe Incorporated Closed nucleic acid structures
WO2013036668A1 (en) 2011-09-06 2013-03-14 Gen-Probe Incorporated Circularized templates for sequencing
AU2012316218B2 (en) 2011-09-26 2016-03-17 Gen-Probe Incorporated Algorithms for sequence determinations
CA2852665A1 (en) 2011-10-17 2013-04-25 Good Start Genetics, Inc. Analysis methods
EP3305400A3 (en) 2011-10-28 2018-06-06 Illumina, Inc. Microarray fabrication system and method
US8637242B2 (en) 2011-11-07 2014-01-28 Illumina, Inc. Integrated sequencing apparatuses and methods of use
US9200274B2 (en) 2011-12-09 2015-12-01 Illumina, Inc. Expanded radix for polymeric tags
BR112014015152A2 (en) 2011-12-21 2017-07-04 Myriad Genetics Inc methods and materials for the assessment of loss of heterozygosity
EP2802666B1 (en) 2012-01-13 2018-09-19 Data2Bio Genotyping by next-generation sequencing
WO2013117595A2 (en) 2012-02-07 2013-08-15 Illumina Cambridge Limited Targeted enrichment and amplification of nucleic acids on a support
US20130217023A1 (en) 2012-02-22 2013-08-22 454 Life Sciences Corporation System And Method For Generation And Use Of Compact Clonally Amplified Products
CA2864481C (en) 2012-02-23 2020-07-14 Dana-Farber Cancer Institute, Inc. Methods for predicting anti-cancer response
EP3309262B1 (en) 2012-02-24 2019-09-25 Bio-Rad Laboratories, Inc. Labeling and sample preparation for sequencing
EP2820129A1 (en) 2012-03-02 2015-01-07 Sequenom, Inc. Methods and processes for non-invasive assessment of genetic variations
NO2694769T3 (en) 2012-03-06 2018-03-03
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
US20130274148A1 (en) 2012-04-11 2013-10-17 Illumina, Inc. Portable genetic detection and analysis system and method
US10227635B2 (en) 2012-04-16 2019-03-12 Molecular Loop Biosolutions, Llc Capture reactions
US9646132B2 (en) 2012-05-11 2017-05-09 Life Technologies Corporation Models for analyzing data from sequencing-by-synthesis operations
US10192024B2 (en) 2012-05-18 2019-01-29 454 Life Sciences Corporation System and method for generation and use of optimal nucleotide flow orders
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
WO2013182645A1 (en) 2012-06-07 2013-12-12 Institut Curie Methods for detecting inactivation of the homologous recombination pathway (brca1/2) in human tumors
US9012022B2 (en) 2012-06-08 2015-04-21 Illumina, Inc. Polymer coatings
US8895249B2 (en) 2012-06-15 2014-11-25 Illumina, Inc. Kinetic exclusion amplification of nucleic acid libraries
US20150167084A1 (en) 2012-07-03 2015-06-18 Sloan Kettering Institute For Cancer Research Quantitative Assessment of Human T-Cell Repertoire Recovery After Allogeneic Hematopoietic Stem Cell Transplantation
US20140093873A1 (en) 2012-07-13 2014-04-03 Sequenom, Inc. Processes and compositions for methylation-based enrichment of fetal nucleic acid from a maternal sample useful for non-invasive prenatal diagnoses
US11155860B2 (en) 2012-07-19 2021-10-26 Oxford Nanopore Technologies Ltd. SSB method
NL2017959B1 (en) 2016-12-08 2018-06-19 Illumina Inc Cartridge assembly
WO2014031157A1 (en) 2012-08-20 2014-02-27 Illumina, Inc. Method and system for fluorescence lifetime based sequencing
US10329608B2 (en) 2012-10-10 2019-06-25 Life Technologies Corporation Methods, systems, and computer readable media for repeat sequencing
EP4592400A3 (en) 2012-10-17 2025-10-29 10x Genomics Sweden AB Methods and product for optimising localised or spatial detection of gene expression in a tissue sample
US9181583B2 (en) 2012-10-23 2015-11-10 Illumina, Inc. HLA typing using selective amplification and sequencing
US9914966B1 (en) 2012-12-20 2018-03-13 Nabsys 2.0 Llc Apparatus and methods for analysis of biomolecules using high frequency alternating current excitation
EP2746405B1 (en) 2012-12-23 2015-11-04 HS Diagnomics GmbH Methods and primer sets for high throughput PCR sequencing
US9683230B2 (en) 2013-01-09 2017-06-20 Illumina Cambridge Limited Sample preparation on a solid support
WO2014108850A2 (en) 2013-01-09 2014-07-17 Yeda Research And Development Co. Ltd. High throughput transcriptome analysis
US10294516B2 (en) 2013-01-18 2019-05-21 Nabsys 2.0 Llc Enhanced probe binding
US9896728B2 (en) 2013-01-29 2018-02-20 Arcticrx Ltd. Method for determining a therapeutic approach for the treatment of age-related macular degeneration (AMD)
US9512422B2 (en) 2013-02-26 2016-12-06 Illumina, Inc. Gel patterned surfaces
EP2964785B8 (en) 2013-03-04 2020-10-21 Fry Laboratories LLC Method and kit for characterizing microorganisms
ES2662598T3 (en) 2013-03-08 2018-04-09 F. Hoffmann-La Roche Ag Blood tests for the detection of EGFR mutations
CA2901545C (en) 2013-03-08 2019-10-08 Oxford Nanopore Technologies Limited Use of spacer elements in a nucleic acid to control movement of a helicase
DK2970951T3 (en) 2013-03-13 2019-05-13 Illumina Inc PROCEDURES FOR NUCLEAR ACID SEQUENCE
DK2969479T3 (en) 2013-03-13 2021-08-02 Illumina Inc Multilayer liquid devices and methods for their manufacture
US11060145B2 (en) 2013-03-13 2021-07-13 Sequenom, Inc. Methods and compositions for identifying presence or absence of hypermethylation or hypomethylation locus
US10308986B2 (en) 2013-03-14 2019-06-04 Children's Medical Center Corporation Cancer diagnosis, treatment selection and treatment
US20140296080A1 (en) 2013-03-14 2014-10-02 Life Technologies Corporation Methods, Systems, and Computer Readable Media for Evaluating Variant Likelihood
EP2971159B1 (en) 2013-03-14 2019-05-08 Molecular Loop Biosolutions, LLC Methods for analyzing nucleic acids
EP2971070B2 (en) 2013-03-14 2021-03-03 Illumina, Inc. Modified polymerases for improved incorporation of nucleotide analogues
WO2014144883A1 (en) 2013-03-15 2014-09-18 The Trustees Of Columbia University In The City Of New York Raman cluster tagged molecules for biological imaging
WO2014139596A1 (en) 2013-03-15 2014-09-18 Illumina Cambridge Limited Modified nucleosides or nucleotides
US9193998B2 (en) 2013-03-15 2015-11-24 Illumina, Inc. Super resolution imaging
CA2909479A1 (en) 2013-05-09 2014-11-13 F. Hoffmann-La Roche Ag Method of determining the fraction of fetal dna in maternal blood using hla markers
WO2014197377A2 (en) 2013-06-03 2014-12-11 Good Start Genetics, Inc. Methods and systems for storing sequence read data
CA2916662C (en) 2013-06-25 2022-03-08 Prognosys Biosciences, Inc. Methods and systems for determining spatial patterns of biological targets in a sample
DK3017065T3 (en) 2013-07-01 2018-11-26 Illumina Inc Catalyst-free Surface functionalization and polymer grafting
US9926597B2 (en) 2013-07-26 2018-03-27 Life Technologies Corporation Control nucleic acid sequences for use in sequencing-by-synthesis and methods for designing the same
EP2840148B1 (en) 2013-08-23 2019-04-03 F. Hoffmann-La Roche AG Methods for nucleic acid amplification
EP2848698A1 (en) 2013-08-26 2015-03-18 F. Hoffmann-La Roche AG System and method for automated nucleic acid amplification
US10508311B2 (en) 2013-08-26 2019-12-17 The Translational Genomics Research Institute Single molecule-overlapping read analysis for minor variant mutation detection in pathogen samples
KR20160081896A (en) 2013-08-30 2016-07-08 일루미나, 인코포레이티드 Manipulation of droplets on hydrophilic or variegated-hydrophilic surfaces
US11901041B2 (en) 2013-10-04 2024-02-13 Bio-Rad Laboratories, Inc. Digital analysis of nucleic acid modification
CN105683980B (en) 2013-10-04 2018-08-24 生命科技股份有限公司 The method and system of effect model stage by stage is established in using the sequencing for terminating chemical substance
EP3058096A1 (en) 2013-10-18 2016-08-24 Good Start Genetics, Inc. Methods for assessing a genomic region of a subject
US10851414B2 (en) 2013-10-18 2020-12-01 Good Start Genetics, Inc. Methods for determining carrier status
CN105874460B (en) 2013-11-01 2018-10-02 精赛恩公司 Identify method, readable medium and the equipment of at least one base of target sequence
CN105849284B (en) * 2013-11-01 2021-08-10 奥基诺公司 Method and apparatus for separating quality levels in sequence data and sequencing longer reads
GB201321123D0 (en) 2013-11-29 2014-01-15 Linea Ab Q Amplification of circular molecules
CA3181696A1 (en) 2013-12-03 2015-06-11 Paul BELITZ Methods and systems for analyzing image data
WO2015086473A1 (en) 2013-12-09 2015-06-18 Institut Curie Methods for detecting inactivation of the homologous recombination pathway (brca1/2) in human tumors
US9944977B2 (en) 2013-12-12 2018-04-17 Raindance Technologies, Inc. Distinguishing rare variations in a nucleic acid sequence from a sample
EP3957750B1 (en) 2013-12-20 2025-01-29 Illumina, Inc. Preserving genomic connectivity information in fragmented genomic dna samples
JP7082860B2 (en) 2013-12-23 2022-06-09 イラミーナ インコーポレーテッド Methods for Structured and Structured Substrate to Improve Detection of Light Radiation
US10537889B2 (en) 2013-12-31 2020-01-21 Illumina, Inc. Addressable flow cell using patterned electrodes
WO2015103367A1 (en) 2013-12-31 2015-07-09 Raindance Technologies, Inc. System and method for detection of rna species
US9677132B2 (en) 2014-01-16 2017-06-13 Illumina, Inc. Polynucleotide modification on solid support
KR102001554B1 (en) 2014-01-16 2019-07-18 일루미나, 인코포레이티드 Amplicon preparation and sequencing on solid supports
HRP20210953T1 (en) 2014-02-18 2021-09-03 Illumina, Inc. Methods and compositions for dna profiling
GB201403096D0 (en) 2014-02-21 2014-04-09 Oxford Nanopore Tech Ltd Sample preparation method
EP3116651B1 (en) 2014-03-11 2020-04-22 Illumina, Inc. Disposable, integrated microfluidic cartridge and methods of making it
EP3736344A1 (en) 2014-03-13 2020-11-11 Sequenom, Inc. Methods and processes for non-invasive assessment of genetic variations
WO2015159292A2 (en) 2014-04-14 2015-10-22 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. A method and kit for determining the tissue or cell origin of dna
WO2015159295A1 (en) 2014-04-17 2015-10-22 Yeda Research And Development Co. Ltd. Methods and kits for analyzing dna binding moieties attached to dna
US11053548B2 (en) 2014-05-12 2021-07-06 Good Start Genetics, Inc. Methods for detecting aneuploidy
CA3219413A1 (en) 2014-05-16 2015-11-19 Illumina, Inc. Nucleic acid synthesis techniques
US20150353989A1 (en) 2014-06-09 2015-12-10 Illumina Cambridge Limited Sample preparation for nucleic acid amplification
CN106715693A (en) 2014-06-13 2017-05-24 伊卢米纳剑桥有限公司 Methods and compositions for preparing sequence libraries
US10829814B2 (en) 2014-06-19 2020-11-10 Illumina, Inc. Methods and compositions for single cell genomics
US10017759B2 (en) 2014-06-26 2018-07-10 Illumina, Inc. Library preparation of tagged nucleic acid
EP3702471B1 (en) 2014-06-27 2025-12-24 Illumina, Inc. Modified polymerases for improved incorporation of nucleotide analogues
CN112430641A (en) 2014-06-30 2021-03-02 亿明达股份有限公司 Methods and compositions using unilateral transposition
US20160017421A1 (en) 2014-07-16 2016-01-21 Roche Molecular Systems Non-invasive early detection of solid organ transplant rejection by quantitative analysis of hla gene amplicons
CA2955382C (en) 2014-07-21 2023-07-18 Illumina, Inc. Polynucleotide enrichment using crispr-cas systems
GB201414098D0 (en) 2014-08-08 2014-09-24 Illumina Cambridge Ltd Modified nucleotide linkers
CA2958801A1 (en) 2014-08-15 2016-02-18 Myriad Genetics, Inc. Methods and materials for assessing homologous recombination deficiency
WO2016025818A1 (en) 2014-08-15 2016-02-18 Good Start Genetics, Inc. Systems and methods for genetic analysis
US9982250B2 (en) 2014-08-21 2018-05-29 Illumina Cambridge Limited Reversible surface functionalization
WO2016040446A1 (en) 2014-09-10 2016-03-17 Good Start Genetics, Inc. Methods for selectively suppressing non-target sequences
WO2016040602A1 (en) 2014-09-11 2016-03-17 Epicentre Technologies Corporation Reduced representation bisulfite sequencing using uracil n-glycosylase (ung) and endonuclease iv
WO2016044233A1 (en) 2014-09-18 2016-03-24 Illumina, Inc. Methods and systems for analyzing nucleic acid sequencing data
JP2017536087A (en) 2014-09-24 2017-12-07 グッド スタート ジェネティクス， インコーポレイテッド Process control to increase the robustness of genetic assays
CN107002051A (en) 2014-09-30 2017-08-01 亿明达股份有限公司 The polymerase through modification for the improved incorporation of nucleotide analog
WO2016060974A1 (en) 2014-10-13 2016-04-21 Life Technologies Corporation Methods, systems, and computer-readable media for accelerated base calling
US10494630B2 (en) * 2014-10-14 2019-12-03 Mgi Tech Co., Ltd. Linker element and method of using same to construct sequencing library
US9897791B2 (en) 2014-10-16 2018-02-20 Illumina, Inc. Optical scanning systems for in situ genetic analysis
RU2736728C2 (en) 2014-10-17 2020-11-19 Иллумина Кембридж Лимитед Transposition with preservation of gene adhesion
WO2016066586A1 (en) 2014-10-31 2016-05-06 Illumina Cambridge Limited Novel polymers and dna copolymer coatings
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
ES2768762T3 (en) 2014-11-05 2020-06-23 Illumina Cambridge Ltd Reduction of DNA damage during sample preparation and sequencing using siderophore chelating agents
CN114438174B (en) 2014-11-11 2025-07-15 伊鲁米那股份有限公司 Polynucleotide amplification using the CRISPR-Cas system
PT3218511T (en) 2014-11-11 2020-07-23 Illumina Cambridge Ltd Methods and arrays for producing and sequencing monoclonal clusters of nucleic acid
WO2016082129A1 (en) * 2014-11-26 2016-06-02 深圳华大基因研究院 Method and reagent for constructing nucleic acid double-linker single-strand cyclic library
CN107002291B (en) * 2014-11-26 2019-03-26 深圳华大智造科技有限公司 A kind of construction method and reagent of double-linker single-stranded circular library of nucleic acid
DK3234187T3 (en) 2014-12-15 2021-05-25 Illumina Inc Method for single molecule placement on a substrate
WO2016112073A1 (en) 2015-01-06 2016-07-14 Good Start Genetics, Inc. Screening for structural variants
CN107406890B (en) 2015-02-10 2023-07-18 亿明达股份有限公司 Methods and compositions for analyzing cellular components
US10576471B2 (en) 2015-03-20 2020-03-03 Illumina, Inc. Fluidics cartridge for use in the vertical or substantially vertical position
CA3225867A1 (en) 2015-03-24 2016-09-29 Illumina, Inc. Methods, carrier assemblies, and systems for imaging samples for biological or chemical analysis
WO2016156845A1 (en) 2015-03-31 2016-10-06 Illumina Cambridge Limited Surface concatamerization of templates
WO2016161085A1 (en) 2015-04-01 2016-10-06 Cedars-Sinai Medical Center Anti-methanogenic lovastatin analogs or derivatives and uses thereof
EP3901282B1 (en) 2015-04-10 2023-06-28 Spatial Transcriptomics AB Spatially distinguished, multiplex nucleic acid analysis of biological specimens
EP3696536A1 (en) 2015-04-14 2020-08-19 Illumina, Inc. A method of manufacturing a substrate and a method of analyzing biomolecules capable of generating light emissions
RU2724998C2 (en) 2015-05-11 2020-06-29 Иллюмина, Инк. Platform for detecting and analyzing therapeutic agents
EP4220645A3 (en) 2015-05-14 2023-11-08 Life Technologies Corporation Barcode sequences, and related systems and methods
WO2016196358A1 (en) 2015-05-29 2016-12-08 Epicentre Technologies Corporation Methods of analyzing nucleic acids
AU2016269785B2 (en) 2015-05-29 2019-02-28 Illumina Cambridge Limited Enhanced utilization of surface primers in clusters
EP3310933B1 (en) 2015-06-17 2020-11-18 The Translational Genomics Research Institute Methods for obtaining biological molecules from a sample
US10632157B2 (en) 2016-04-15 2020-04-28 Ascus Biosciences, Inc. Microbial compositions and methods of use for improving fowl production
US9938558B2 (en) 2015-06-25 2018-04-10 Ascus Biosciences, Inc. Methods, apparatuses, and systems for analyzing microorganism strains from complex heterogeneous communities, predicting and identifying functional relationships and interactions thereof, and selecting and synthesizing microbial ensembles based thereon
PY1639434A (en) 2015-06-25 2018-08-01 Ascus Biosciences Inc PROCEDURES, APPARATUS AND SYSTEMS FOR THE ANALYSIS OF MICROORGANISM STRAINS FROM COMPLEX AND HETEROGENEOUS COMMUNITIES. PREDICTION AND IDENTIFICATION OF RELATIONSHIPS..//.
US10851399B2 (en) 2015-06-25 2020-12-01 Native Microbials, Inc. Methods, apparatuses, and systems for microorganism strain analysis of complex heterogeneous communities, predicting and identifying functional relationships and interactions thereof, and selecting and synthesizing microbial ensembles based thereon
EP3878974A1 (en) 2015-07-06 2021-09-15 Illumina Cambridge Limited Sample preparation for nucleic acid amplification
US10808282B2 (en) 2015-07-07 2020-10-20 Illumina, Inc. Selective surface patterning via nanoimprinting
EP3325648B1 (en) 2015-07-17 2023-03-29 Illumina, Inc. Polymer sheets for sequencing applications
CA3176469A1 (en) 2015-07-27 2017-02-02 Illumina, Inc. Spatial mapping of nucleic acid sequence information
WO2017021961A1 (en) 2015-08-04 2017-02-09 Yeda Research And Development Co. Ltd. Methods of screening for riboswitches and attenuators
DK3334839T3 (en) 2015-08-14 2021-04-26 Illumina Inc SYSTEMS AND PROCEDURES USING MAGNETIC REACTIVE SENSORS FOR DETERMINING A GENETIC PROPERTY
HK1253910A1 (en) 2015-09-02 2019-07-05 伊卢米纳剑桥有限公司 Systems and methods of improving droplet operations in fluidic systems
US10647981B1 (en) 2015-09-08 2020-05-12 Bio-Rad Laboratories, Inc. Nucleic acid library generation methods and compositions
US10450598B2 (en) 2015-09-11 2019-10-22 Illumina, Inc. Systems and methods for obtaining a droplet having a designated concentration of a substance-of-interest
CN109072300B (en) 2015-12-17 2023-01-31 伊路敏纳公司 Differentiating methylation levels in complex biological samples
MX392304B (en) 2016-01-07 2025-03-24 Native Microbials Inc METHODS FOR IMPROVING MILK PRODUCTION BY ADMINISTRATION OF MICROBIAL CONSORTIUMS.
PL3377226T3 (en) 2016-03-28 2021-07-26 Illumina, Inc. Multi-plane microarrays
US10961573B2 (en) 2016-03-28 2021-03-30 Boreal Genomics, Inc. Linked duplex target capture
WO2017168332A1 (en) 2016-03-28 2017-10-05 Boreal Genomics, Inc. Linked duplex target capture
WO2017177017A1 (en) 2016-04-07 2017-10-12 Omniome, Inc. Methods of quantifying target nucleic acids and identifying sequence variants
MX391417B (en) 2016-04-15 2025-03-21 Native Microbials Inc METHODS FOR IMPROVING AGRICULTURAL POULTRY PRODUCTION BY ADMINISTRATION OF MICROBIAL CONSORTIUMS OR PURIFIED STRAINS THEREOF.
US10619205B2 (en) 2016-05-06 2020-04-14 Life Technologies Corporation Combinatorial barcode sequences, and related systems and methods
DK3455372T3 (en) 2016-05-11 2020-01-06 Illumina Inc Polynucleotide Enrichment and Amplification Using Argonauts
GB201609220D0 (en) 2016-05-25 2016-07-06 Oxford Nanopore Tech Ltd Method
EP3904514A1 (en) 2016-07-22 2021-11-03 Oregon Health & Science University Single cell whole genome libraries and combinatorial indexing methods of making thereof
US20190203270A1 (en) 2016-07-24 2019-07-04 Yeda Research And Development Co., Ltd. Methods and kits for analyzing dna binding moieties attached to dna
WO2018064116A1 (en) 2016-09-28 2018-04-05 Illumina, Inc. Methods and systems for data compression
FI3522713T3 (en) 2016-10-03 2023-01-13 Fluorescent detection of amines and hydrazines and assaying methods thereof
MY194951A (en) 2016-10-14 2022-12-28 Illumina Inc Cartridge assembly
GB201619458D0 (en) 2016-11-17 2017-01-04 Spatial Transcriptomics Ab Method for spatial tagging and analysing nucleic acids in a biological specimen
US11268137B2 (en) 2016-12-09 2022-03-08 Boreal Genomics, Inc. Linked ligation
CA3048246A1 (en) 2016-12-28 2018-07-05 Ascus Biosciences, Inc. Methods, apparatuses, and systems for analyzing complete microorganism strains in complex heterogeneous communities, determining functional relationships and interactions thereof, and identifying and synthesizing bioreactive modificators based thereon
AU2017388535A1 (en) 2016-12-28 2019-07-25 Native Microbials, Inc. Methods, apparatuses, and systems for microorganism strain analysis of complex heterogeneous communities with tracer analytics, determination of functional relationships and interactions thereof, and synthesis of microbial ensembles including dosed microbial ensembles and inoculative microbial ensembles
ES3026859T3 (en) 2017-01-04 2025-06-12 Mgi Tech Co Ltd Stepwise sequencing by non-labeled reversible terminators or natural nucleotides
GB201704754D0 (en) 2017-01-05 2017-05-10 Illumina Inc Kinetic exclusion amplification of nucleic acid libraries
US11150179B2 (en) 2017-01-06 2021-10-19 Illumina, Inc. Phasing correction
WO2018128544A1 (en) 2017-01-06 2018-07-12 Agendia N.V. Biomarkers for selecting patient groups, and uses thereof.
BR112019014042A2 (en) 2017-01-17 2020-02-04 Illumina, Inc. determination of oncogenic splice variant
CN110249059B (en) 2017-01-20 2021-04-13 欧姆尼欧美公司 Allele-specific capture of nucleic acids
CA3050852C (en) 2017-01-20 2021-03-09 Omniome, Inc. Genotyping by polymerase binding
GB201701688D0 (en) 2017-02-01 2017-03-15 Illumia Inc System and method with fiducials in non-recliner layouts
GB201701689D0 (en) 2017-02-01 2017-03-15 Illumia Inc System and method with fiducials of non-closed shapes
CN109414673B (en) 2017-02-01 2021-09-07 伊鲁米那股份有限公司 Systems and methods with fiducials responsive to multiple excitation frequencies
GB201701686D0 (en) 2017-02-01 2017-03-15 Illunina Inc System & method with fiducials having offset layouts
US11492666B2 (en) 2017-02-15 2022-11-08 Pacific Biosciences Of California, Inc. Distinguishing sequences by detecting polymerase dissociation
EP3494235A1 (en) 2017-02-17 2019-06-12 Stichting VUmc Swarm intelligence-enhanced diagnosis and therapy selection for cancer using tumor- educated platelets
US20200377935A1 (en) 2017-03-24 2020-12-03 Life Technologies Corporation Polynucleotide adapters and methods of use thereof
WO2018187013A1 (en) 2017-04-04 2018-10-11 Omniome, Inc. Fluidic apparatus and methods useful for chemical and biological reactions
EP3913053A1 (en) 2017-04-23 2021-11-24 Illumina Cambridge Limited Compositions and methods for improving sample identification in indexed nucleic acid libraries
UY37708A (en) 2017-04-28 2018-10-31 Ascus Biosciences Inc METHODS TO COMPLEMENT DIETS WITH HIGH CONSUMPTION OF GRAINS AND / OR HIGH ENERGY VALUE IN RUMINANTS, THROUGH THE ADMINISTRATION OF A SYNTHETIC BIOENSAMBLE OF MICROBIES, OR PURIFIED VINTAGES OF THE SAME
DK3635136T3 (en) 2017-06-07 2022-01-10 Univ Oregon Health & Science SINGLE CELL WEEKEND LIBRARIES FOR METHYLATION SEQUENCE
ES3055990T3 (en) 2017-06-20 2026-02-17 Illumina Inc Methods for addressing inefficiencies in amplification reactions
WO2019012542A1 (en) 2017-07-13 2019-01-17 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Detecting tissue-specific dna
EP3652344A1 (en) 2017-07-13 2020-05-20 Yissum Research and Development Company of the Hebrew University of Jerusalem Ltd. Dna targets as tissue-specific methylation markers
EP3662482A1 (en) 2017-07-31 2020-06-10 Illumina Inc. Sequencing system with multiplexed biological sample aggregation
WO2019023924A1 (en) 2017-08-01 2019-02-07 Helitec Limited Methods of enriching and determining target nucleotide sequences
SG11201911869XA (en) 2017-08-01 2020-01-30 Illumina Inc Spatial indexing of genetic material and library preparation using hydrogel beads and flow cells
EP3662084B1 (en) 2017-08-01 2024-10-30 Illumina, Inc. Hydrogel beads for nucleotide sequencing
EP4553172A3 (en) * 2017-08-01 2025-06-25 MGI Tech Co., Ltd. Nucleic acid sequencing method
WO2019035897A1 (en) 2017-08-15 2019-02-21 Omniome, Inc. Scanning apparatus and methods useful for detection of chemical and biological analytes
UY37939A (en) 2017-10-18 2019-04-30 Ascus Biosciences Inc INCREASE IN CORRAL BIRD PRODUCTION THROUGH THE ADMINISTRATION OF A SYNTHETIC BIO-ASSEMBLY OF MICROBES OR PURIFIED VINTAGES OF THE SAME
CA3067140C (en) 2018-02-13 2023-03-21 Illumina, Inc. Dna sequencing using hydrogel beads
CN110832086A (en) 2018-04-02 2020-02-21 伊鲁米那股份有限公司 Compositions and methods for making controls for sequence-based genetic testing
WO2019203986A1 (en) 2018-04-19 2019-10-24 Omniome, Inc. Improving accuracy of base calls in nucleic acid sequencing methods
SG11201911961RA (en) 2018-04-20 2020-01-30 Illumina Inc Methods of encapsulating single cells, the encapsulated cells and uses thereof
GB201807793D0 (en) 2018-05-14 2018-06-27 Oxford Nanopore Tech Ltd Method
IL271454B2 (en) 2018-05-17 2025-04-01 Illumina Inc High-throughput single-cell sequencing with reduced amplification bias
DK3810774T3 (en) 2018-06-04 2023-12-11 Illumina Inc HIGH-THROUGH-PUT SINGLE CELL TRANSCRIPTOME LIBRARIES AND METHODS OF PREPARATION AND USE
WO2020022891A2 (en) 2018-07-26 2020-01-30 Stichting Vumc Biomarkers for atrial fibrillation
US12098419B2 (en) 2018-08-23 2024-09-24 Ncan Genomics, Inc. Linked target capture and ligation
US11519033B2 (en) 2018-08-28 2022-12-06 10X Genomics, Inc. Method for transposase-mediated spatial tagging and analyzing genomic DNA in a biological sample
WO2020047005A1 (en) 2018-08-28 2020-03-05 10X Genomics, Inc. Resolving spatial arrays
US20210332424A1 (en) 2018-08-28 2021-10-28 10X Genomics, Inc. Methods of generating an array
EP3853358A1 (en) 2018-09-17 2021-07-28 Omniome, Inc. Engineered polymerases for improved sequencing
WO2020086843A1 (en) 2018-10-26 2020-04-30 Illumina, Inc. Modulating polymer beads for dna processing
EP3874036A1 (en) 2018-10-31 2021-09-08 Illumina, Inc. Polymerases, compositions, and methods of use
NL2022043B1 (en) 2018-11-21 2020-06-03 Akershus Univ Hf Tagmentation-Associated Multiplex PCR Enrichment Sequencing
EP3888021B1 (en) 2018-11-30 2024-02-21 Caris MPI, Inc. Next-generation molecular profiling
CN112867801B (en) 2018-11-30 2024-07-12 Illumina公司 Analyze multiple analytes using a single assay
EP3891304A1 (en) 2018-12-04 2021-10-13 Omniome, Inc. Mixed-phase fluids for nucleic acid sequencing and other analytical assays
SG11202012762WA (en) 2018-12-05 2021-01-28 Illumina Cambridge Ltd Methods and compositions for cluster generation by bridge amplification
WO2020117968A2 (en) 2018-12-05 2020-06-11 Illumina, Inc. Polymerases, compositions, and methods of use
WO2020123316A2 (en) 2018-12-10 2020-06-18 10X Genomics, Inc. Methods for determining a location of a biological analyte in a biological sample
US12529094B2 (en) 2018-12-10 2026-01-20 10X Genomics, Inc. Imaging system hardware
GB201820300D0 (en) 2018-12-13 2019-01-30 10X Genomics Inc Method for spatial tagging and analysing genomic DNA in a biological specimen
GB201820341D0 (en) 2018-12-13 2019-01-30 10X Genomics Inc Method for transposase-mediated spatial tagging and analysing genomic DNA in a biological specimen
JP7587425B2 (en) 2018-12-18 2024-11-20 イルミナ ケンブリッジ リミテッド Methods and compositions for paired-end sequencing using a single surface primer
DK3899037T3 (en) 2018-12-19 2023-11-06 Illumina Inc METHODS OF IMPROVING POLYNUCLEOTIDE CLUSTER CLONALITY PRIORITY
EP3899032A2 (en) 2018-12-20 2021-10-27 Omniome, Inc. Temperature control for analysis of nucleic acids and other analytes
US11293061B2 (en) 2018-12-26 2022-04-05 Illumina Cambridge Limited Sequencing methods using nucleotides with 3′ AOM blocking group
EP4538389A3 (en) 2019-01-03 2025-07-30 Ncan Genomics, Inc. Linked target capture
US11649485B2 (en) 2019-01-06 2023-05-16 10X Genomics, Inc. Generating capture probes for spatial analysis
US11926867B2 (en) 2019-01-06 2024-03-12 10X Genomics, Inc. Generating capture probes for spatial analysis
CN114174531A (en) 2019-02-28 2022-03-11 10X基因组学有限公司 Profiling of biological analytes with spatially barcoded oligonucleotide arrays
BR112021006234A2 (en) 2019-03-01 2021-09-28 Illumina, Inc. HIGH PERFORMANCE SINGLE CELL AND SINGLE CORE LIBRARIES AND METHODS OF PREPARATION AND USE
EP3938538A1 (en) 2019-03-15 2022-01-19 10X Genomics, Inc. Methods for using spatial arrays for single cell sequencing
EP3887542A1 (en) 2019-03-22 2021-10-06 10X Genomics, Inc. Three-dimensional spatial analysis
WO2020243579A1 (en) 2019-05-30 2020-12-03 10X Genomics, Inc. Methods of detecting spatial heterogeneity of a biological sample
US11644406B2 (en) 2019-06-11 2023-05-09 Pacific Biosciences Of California, Inc. Calibrated focus sensing
MX2021007803A (en) 2019-07-12 2021-08-11 Illumina Cambridge Ltd Compositions and methods for preparing nucleic acid sequencing libraries using crispr/cas9 immobilized on a solid support.
US10656368B1 (en) 2019-07-24 2020-05-19 Omniome, Inc. Method and system for biological imaging using a wide field objective lens
US20220290245A1 (en) 2019-09-11 2022-09-15 The United States Of America, As Represented By The Secretary, Department Of Health And Human Servic Cancer detection and classification
EP4468252A3 (en) 2019-10-01 2025-02-19 10x Genomics, Inc. Systems and methods for identifying morphological patterns in tissue samples
EP4045683B1 (en) 2019-10-18 2025-02-19 Pacific Biosciences of California, Inc. Methods for capping nucleic acids
BR112021018125A2 (en) 2019-10-25 2021-11-16 Illumina Cambridge Ltd Methods for generating an asymmetric closed-end double-stranded nucleic acid template, for generating an asymmetrical double-stranded nucleic acid template from tagged DNA, and sequencing tagged DNA
US12157124B2 (en) 2019-11-06 2024-12-03 10X Genomics, Inc. Imaging system hardware
EP4055155A1 (en) 2019-11-08 2022-09-14 Pacific Biosciences of California, Inc. Engineered polymerases for improved sequencing by binding
WO2021091611A1 (en) 2019-11-08 2021-05-14 10X Genomics, Inc. Spatially-tagged analyte capture agents for analyte multiplexing
WO2021092433A2 (en) 2019-11-08 2021-05-14 10X Genomics, Inc. Enhancing specificity of analyte binding
WO2021102003A1 (en) 2019-11-18 2021-05-27 10X Genomics, Inc. Systems and methods for tissue classification
AU2020388573A1 (en) 2019-11-21 2022-06-23 10X Genomics, Inc, Spatial analysis of analytes
EP4062372B1 (en) 2019-11-22 2024-05-08 10X Genomics, Inc. Systems and methods for spatial analysis of analytes using fiducial alignment
DE202019106694U1 (en) 2019-12-02 2020-03-19 Omniome, Inc. System for sequencing nucleic acids in fluid foam
CA3163319A1 (en) 2019-12-02 2021-06-10 Caris Mpi, Inc. Pan-cancer platinum response predictor
WO2021118349A1 (en) 2019-12-10 2021-06-17 Prinses Máxima Centrum Voor Kinderoncologie B.V. Methods of typing germ cell tumors
JP2023508792A (en) 2019-12-19 2023-03-06 イルミナ インコーポレイテッド HIGH THROUGHPUT SINGLE-CELL LIBRARIES AND METHODS OF MAKING AND USING
ES2982420T3 (en) 2019-12-23 2024-10-16 10X Genomics Inc Methods for spatial analysis using RNA-templated ligation
EP4081656A1 (en) 2019-12-23 2022-11-02 10X Genomics, Inc. Compositions and methods for using fixed biological samples in partition-based assays
CN115135984B (en) 2019-12-23 2025-12-23 10X基因组学有限公司 Reversible fixation reagents and their usage
US12365942B2 (en) 2020-01-13 2025-07-22 10X Genomics, Inc. Methods of decreasing background on a spatial array
US12405264B2 (en) 2020-01-17 2025-09-02 10X Genomics, Inc. Electrophoretic system and method for analyte capture
US11732299B2 (en) 2020-01-21 2023-08-22 10X Genomics, Inc. Spatial assays with perturbed cells
US11702693B2 (en) 2020-01-21 2023-07-18 10X Genomics, Inc. Methods for printing cells and generating arrays of barcoded cells
US20210230681A1 (en) 2020-01-24 2021-07-29 10X Genomics, Inc. Methods for spatial analysis using proximity ligation
US11821035B1 (en) 2020-01-29 2023-11-21 10X Genomics, Inc. Compositions and methods of making gene expression libraries
US12076701B2 (en) 2020-01-31 2024-09-03 10X Genomics, Inc. Capturing oligonucleotides in spatial transcriptomics
US12110548B2 (en) 2020-02-03 2024-10-08 10X Genomics, Inc. Bi-directional in situ analysis
US12110541B2 (en) 2020-02-03 2024-10-08 10X Genomics, Inc. Methods for preparing high-resolution spatial arrays
US11898205B2 (en) 2020-02-03 2024-02-13 10X Genomics, Inc. Increasing capture efficiency of spatial assays
US12112833B2 (en) 2020-02-04 2024-10-08 10X Genomics, Inc. Systems and methods for index hopping filtering
EP4100161B1 (en) 2020-02-04 2024-07-10 Pacific Biosciences of California, Inc. Flow cells and methods for their use
US11732300B2 (en) 2020-02-05 2023-08-22 10X Genomics, Inc. Increasing efficiency of spatial analysis in a biological sample
WO2021158925A1 (en) 2020-02-07 2021-08-12 10X Genomics, Inc. Quantitative and automated permeabilization performance evaluation for spatial transcriptomics
WO2021163212A1 (en) 2020-02-10 2021-08-19 Native Microbials, Inc. Microbial compositions and methods of use for canine enteropathy and dysbiosis
US11835462B2 (en) 2020-02-11 2023-12-05 10X Genomics, Inc. Methods and compositions for partitioning a biological sample
CN115428088A (en) 2020-02-13 2022-12-02 10X基因组学有限公司 Systems and methods for joint interactive visualization of gene expression and DNA chromatin accessibility
US12281357B1 (en) 2020-02-14 2025-04-22 10X Genomics, Inc. In situ spatial barcoding
US12399123B1 (en) 2020-02-14 2025-08-26 10X Genomics, Inc. Spatial targeting of analytes
EP4484571A3 (en) 2020-02-21 2025-03-19 10X Genomics, Inc. Methods and compositions for integrated in situ spatial assay
US11891654B2 (en) 2020-02-24 2024-02-06 10X Genomics, Inc. Methods of making gene expression libraries
US11926863B1 (en) 2020-02-27 2024-03-12 10X Genomics, Inc. Solid state single cell method for analyzing fixed biological cells
CA3173798A1 (en) 2020-03-03 2021-09-10 Pacific Biosciences Of California, Inc. Methods and compositions for sequencing double stranded nucleic acids
US11768175B1 (en) 2020-03-04 2023-09-26 10X Genomics, Inc. Electrophoretic methods for spatial analysis
US12188085B2 (en) 2020-03-05 2025-01-07 10X Genomics, Inc. Three-dimensional spatial transcriptomics with sequencing readout
WO2021178893A2 (en) 2020-03-06 2021-09-10 Singular Genomics Systems, Inc. Linked paired strand sequencing
ES2985378T3 (en) 2020-03-30 2024-11-05 Illumina Inc Methods and compositions for preparing nucleic acid libraries
EP4139485B1 (en) 2020-04-22 2023-09-06 10X Genomics, Inc. Methods for spatial analysis using targeted rna depletion
EP4143344A1 (en) 2020-04-27 2023-03-08 Agendia N.V. Treatment of her2 negative, mammaprint high risk 2 breast cancer
EP4150065A2 (en) 2020-05-12 2023-03-22 Illumina Inc Generating nucleic acids with modified bases using recombinant terminal deoxynucleotidyl transferase
US12416603B2 (en) 2020-05-19 2025-09-16 10X Genomics, Inc. Electrophoresis cassettes and instrumentation
EP4153776B1 (en) 2020-05-22 2025-03-05 10X Genomics, Inc. Spatial analysis to detect sequence variants
EP4414459B1 (en) 2020-05-22 2025-09-03 10X Genomics, Inc. Simultaneous spatio-temporal measurement of gene expression and cellular activity
WO2021242834A1 (en) 2020-05-26 2021-12-02 10X Genomics, Inc. Method for resetting an array
EP4600376A3 (en) 2020-06-02 2025-10-22 10X Genomics, Inc. Spatial transcriptomics for antigen-receptors
US12265079B1 (en) 2020-06-02 2025-04-01 10X Genomics, Inc. Systems and methods for detecting analytes from captured single biological particles
AU2021283174A1 (en) 2020-06-02 2023-01-05 10X Genomics, Inc. Nucleic acid library methods
US12031177B1 (en) 2020-06-04 2024-07-09 10X Genomics, Inc. Methods of enhancing spatial resolution of transcripts
EP4421186B1 (en) 2020-06-08 2025-08-13 10X Genomics, Inc. Methods of determining a surgical margin and methods of use thereof
WO2021252375A1 (en) 2020-06-08 2021-12-16 The Broad Institute, Inc. Single cell combinatorial indexing from amplified nucleic acids
WO2021252617A1 (en) 2020-06-09 2021-12-16 Illumina, Inc. Methods for increasing yield of sequencing libraries
WO2021252747A1 (en) 2020-06-10 2021-12-16 1Ox Genomics, Inc. Fluid delivery methods
EP4165207B1 (en) 2020-06-10 2024-09-25 10X Genomics, Inc. Methods for determining a location of an analyte in a biological sample
US12435363B1 (en) 2020-06-10 2025-10-07 10X Genomics, Inc. Materials and methods for spatial transcriptomics
US12522877B2 (en) 2020-06-17 2026-01-13 The Translational Genomics Research Institute Early detection of drug-resistant mycobacterium tuberculosis
US11787831B2 (en) 2020-06-22 2023-10-17 Illumina Cambridge Limited Nucleosides and nucleotides with 3′ acetal blocking group
ES2994976T3 (en) 2020-06-25 2025-02-05 10X Genomics Inc Spatial analysis of dna methylation
US12168801B1 (en) 2020-07-02 2024-12-17 10X Genomics, Inc. Hybrid/capture probe designs for full-length cDNA
US11761038B1 (en) 2020-07-06 2023-09-19 10X Genomics, Inc. Methods for identifying a location of an RNA in a biological sample
US11981960B1 (en) 2020-07-06 2024-05-14 10X Genomics, Inc. Spatial analysis utilizing degradable hydrogels
US12209280B1 (en) 2020-07-06 2025-01-28 10X Genomics, Inc. Methods of identifying abundance and location of an analyte in a biological sample using second strand synthesis
CN116018412A (en) 2020-07-08 2023-04-25 Illumina公司 Beads as transposome vectors
WO2022015600A2 (en) 2020-07-13 2022-01-20 Singular Genomics Systems, Inc. Methods of sequencing complementary polynucleotides
US11492662B2 (en) 2020-08-06 2022-11-08 Singular Genomics Systems, Inc. Methods for in situ transcriptomics and proteomics
CN116171330A (en) 2020-08-06 2023-05-26 Illumina公司 Preparation of RNA and DNA Sequencing Libraries Using Bead-Linked Transposomes
US12553898B1 (en) 2020-08-10 2026-02-17 10X Genomics, Inc. Fluorescent hybridization of antibody-oligonucleotide for multiplexing and signal amplification
EP4200416A1 (en) 2020-08-18 2023-06-28 Illumina, Inc. Sequence-specific targeted transposition and selection and sorting of nucleic acids
US11981958B1 (en) 2020-08-20 2024-05-14 10X Genomics, Inc. Methods for spatial analysis using DNA capture
US12580044B1 (en) 2020-09-02 2026-03-17 10X Genomics, Inc. Systems and methods for identifying cells that are antigen-specific for an immunogenic feature
MX2023001400A (en) 2020-09-11 2023-04-25 Illumina Cambridge Ltd Methods of enriching a target sequence from a sequencing library using hairpin adaptors.
WO2022061150A2 (en) 2020-09-18 2022-03-24 10X Geonomics, Inc. Sample handling apparatus and image registration methods
US11926822B1 (en) 2020-09-23 2024-03-12 10X Genomics, Inc. Three-dimensional spatial analysis
WO2022087485A1 (en) 2020-10-22 2022-04-28 Singular Genomics Systems, Inc. Nucleic acid circularization and amplification on a surface
US11827935B1 (en) 2020-11-19 2023-11-28 10X Genomics, Inc. Methods for spatial analysis using rolling circle amplification and detection probes
EP4729631A2 (en) 2020-12-21 2026-04-22 10X Genomics, Inc. Methods, compositions, and systems for capturing probes and/or barcodes
MX2023009046A (en) 2021-02-04 2023-08-10 Illumina Inc GENERATION OF EXTENDED INDEXED LINKED READING IN GLOBES UNITED WITH TRANSPOSOMES.
EP4251770A4 (en) 2021-02-08 2024-05-29 Singular Genomics Systems, Inc. Methods and compositions for sequencing complementary polynucleotides
US20240043915A1 (en) 2021-02-13 2024-02-08 The General Hospital Corporation Methods and compositions for in situ macromolecule detection and uses thereof
EP4421491A3 (en) 2021-02-19 2024-11-27 10X Genomics, Inc. Method of using a modular assay support device
US11884977B2 (en) 2021-03-12 2024-01-30 Singular Genomics Systems, Inc. Nanoarrays and methods of use thereof
EP4263868A4 (en) 2021-03-12 2024-11-27 Singular Genomics Systems, Inc. NANOARRAYS AND METHODS OF USE THEREOF
AU2022238446A1 (en) 2021-03-18 2023-09-07 10X Genomics, Inc. Multiplex capture of gene and protein expression from a biological sample
WO2022204032A1 (en) 2021-03-22 2022-09-29 Illumina Cambridge Limited Methods for improving nucleic acid cluster clonality
JP2024513187A (en) 2021-03-29 2024-03-22 イルミナ インコーポレイテッド Compositions and methods for assessing DNA damage and normalizing amplicon size bias in libraries
WO2022212269A1 (en) 2021-03-29 2022-10-06 Illumina, Inc. Improved methods of library preparation
CA3211172A1 (en) 2021-03-31 2022-10-06 Illumina, Inc. Methods of preparing directional tagmentation sequencing libraries using transposon-based technology with unique molecular identifiers for error correction
JP7719206B2 (en) 2021-04-02 2025-08-05 イルミナ インコーポレイテッド Nucleotide for sequencing - Machine learning model for detecting bubbles in specimen slides
EP4305196B1 (en) 2021-04-14 2025-04-02 10X Genomics, Inc. Methods of measuring mislocalization of an analyte
WO2022232308A1 (en) 2021-04-27 2022-11-03 Singular Genomics Systems, Inc. High density sequencing and multiplexed priming
EP4334706A4 (en) 2021-05-05 2025-08-06 Singular Genomics Systems Inc Multiomic analysis device and methods of use thereof
EP4320271B1 (en) 2021-05-06 2025-03-19 10X Genomics, Inc. Methods for increasing resolution of spatial analysis
EP4334475A1 (en) 2021-05-07 2024-03-13 Agendia N.V. Endocrine treatment of hormone receptor positive breast cancer typed as having a low risk of recurrence
KR20240009435A (en) 2021-05-20 2024-01-22 일루미나, 인코포레이티드 Compositions and methods for sequencing by synthesis
WO2022256503A1 (en) 2021-06-03 2022-12-08 10X Genomics, Inc. Methods, compositions, kits, and systems for enhancing analyte capture for spatial analysis
WO2023278966A1 (en) 2021-06-29 2023-01-05 Illumina, Inc. Machine-learning model for generating confidence classifications for genomic coordinates
BR112023026615A2 (en) 2021-06-29 2024-03-05 Illumina Inc SIGNAL-TO-NOISE RATIO METRIC FOR DETERMINING NUCLEOTIDE BASE CALLING AND BASE CALLING QUALITY
US20230021577A1 (en) 2021-07-23 2023-01-26 Illumina Software, Inc. Machine-learning model for recalibrating nucleotide-base calls
US12553805B2 (en) 2021-08-02 2026-02-17 10X Genomics, Inc. Methods of preserving a biological sample
US12077789B2 (en) 2021-08-14 2024-09-03 Illumina, Inc. Polymerases, compositions, and methods of use
CN117881795A (en) 2021-08-17 2024-04-12 因美纳有限公司 Methods and compositions for identifying methylated cytosine
WO2023023638A1 (en) 2021-08-20 2023-02-23 Singular Genomics Systems, Inc. Chemical and thermal assisted nucleic acid amplification methods
CN118103750A (en) 2021-08-31 2024-05-28 伊鲁米纳公司 Flow cell with enhanced pore imaging resolution
WO2023034489A1 (en) 2021-09-01 2023-03-09 10X Genomics, Inc. Methods, compositions, and kits for blocking a capture probe on a spatial array
JP2024535663A (en) 2021-09-17 2024-10-02 イルミナ インコーポレイテッド Automatic Identification of Sources of Faults from Base Call Error Patterns in Nucleotide Sequencing
EP4405954A1 (en) 2021-09-21 2024-07-31 Illumina, Inc. A graph reference genome and base-calling approach using imputed haplotypes
WO2023076832A1 (en) 2021-10-25 2023-05-04 Singular Genomics Systems, Inc. Manipulating and detecting biological samples
EP4422792A4 (en) 2021-10-26 2025-12-17 Singular Genomics Systems Inc TARGETED MULTIPLEXED POLYNUCLEOTIDE AMPLIFICATION
WO2023085932A1 (en) 2021-11-10 2023-05-19 Omnigen B.V. Prediction of response following folfirinox treatment in cancer patients
WO2023086880A1 (en) 2021-11-10 2023-05-19 10X Genomics, Inc. Methods, compositions, and kits for determining the location of an analyte in a biological sample
WO2023102118A2 (en) 2021-12-01 2023-06-08 10X Genomics, Inc. Methods, compositions, and systems for improved in situ detection of analytes and spatial analysis
WO2023102354A1 (en) 2021-12-02 2023-06-08 Illumina Software, Inc. Generating cluster-specific-signal corrections for determining nucleotide-base calls
WO2023122033A1 (en) 2021-12-20 2023-06-29 10X Genomics, Inc. Self-test for pathology/histology slide imaging device
WO2023122363A1 (en) 2021-12-23 2023-06-29 Illumina Software, Inc. Dynamic graphical status summaries for nucelotide sequencing
US20230215515A1 (en) 2021-12-23 2023-07-06 Illumina Software, Inc. Facilitating secure execution of external workflows for genomic sequencing diagnostics
US20230207050A1 (en) 2021-12-28 2023-06-29 Illumina Software, Inc. Machine learning model for recalibrating nucleotide base calls corresponding to target variants
EP4457814A1 (en) 2021-12-29 2024-11-06 Illumina, Inc. Automatically switching variant analysis model versions for genomic analysis applications
WO2023126457A1 (en) 2021-12-29 2023-07-06 Illumina Cambridge Ltd. Methods of nucleic acid sequencing using surface-bound primers
CA3223362A1 (en) 2022-01-20 2023-07-27 Xiaolin Wu Methods of detecting methylcytosine and hydroxymethylcytosine by sequencing
US20230313271A1 (en) 2022-02-25 2023-10-05 Illumina, Inc. Machine-learning models for detecting and adjusting values for nucleotide methylation levels
JP2025507713A (en) 2022-02-25 2025-03-21 イルミナ インコーポレイテッド Calibration Sequences for Nucleotide Sequencing
US20250188521A1 (en) 2022-03-10 2025-06-12 Singular Genomics Systems, Inc. Nucleic acid delivery scaffolds
AU2023248405A1 (en) 2022-04-07 2024-01-04 Illumina, Inc. Altered cytidine deaminases and methods of use
WO2023212601A1 (en) 2022-04-26 2023-11-02 Illumina, Inc. Machine-learning models for selecting oligonucleotide probes for array technologies
US20230368866A1 (en) 2022-05-10 2023-11-16 Illumina Software, Inc. Adaptive neural network for nucelotide sequencing
EP4526471A1 (en) 2022-05-19 2025-03-26 Agendia N.V. Dna repair signature and prediction of response following cancer therapy
US20260049359A1 (en) 2022-05-19 2026-02-19 Agendia N.V. Prediction of response to immune therapy in breast cancer patients
KR20250034034A (en) 2022-06-24 2025-03-10 일루미나, 인코포레이티드 How to improve split-read alignment by intelligently identifying and scoring candidate split groups
US20260011405A1 (en) 2022-06-27 2026-01-08 Illumina, Inc. Human leukocyte antigen (hla) genotyping
CA3260482A1 (en) 2022-06-27 2024-01-04 Illumina, Inc. Generating and implementing a structural variation graph genome
CA3260497A1 (en) 2022-06-27 2024-01-04 Illumina, Inc. Accelerators for a genotype imputation model
WO2024015962A1 (en) 2022-07-15 2024-01-18 Pacific Biosciences Of California, Inc. Blocked asymmetric hairpin adaptors
US20240038327A1 (en) 2022-07-26 2024-02-01 Illumina Software, Inc. Rapid single-cell multiomics processing using an executable file
EP4573217A1 (en) 2022-08-18 2025-06-25 Yissum Research Development Company of the Hebrew University of Jerusalem Ltd. A method for determining the tissue or cell of origin of dna
WO2024039516A1 (en) 2022-08-19 2024-02-22 Illumina, Inc. Third dna base pair site-specific dna detection
CN118974830A (en) 2022-09-29 2024-11-15 因美纳有限公司 Target variant reference set for imputing target variants
US20240141427A1 (en) 2022-09-30 2024-05-02 Illumina, Inc. Polymerases, compositions, and methods of use
EP4594482A1 (en) 2022-09-30 2025-08-06 Illumina, Inc. Cytidine deaminases and methods of use in mapping modified cytosine nucleotides
WO2024073519A1 (en) 2022-09-30 2024-04-04 Illumina, Inc. Machine-learning model for refining structural variant calls
WO2024069581A1 (en) 2022-09-30 2024-04-04 Illumina Singapore Pte. Ltd. Helicase-cytidine deaminase complexes and methods of use
WO2024073043A1 (en) 2022-09-30 2024-04-04 Illumina, Inc. Methods of using cpg binding proteins in mapping modified cytosine nucleotides
KR20250081825A (en) 2022-10-05 2025-06-05 일루미나, 인코포레이티드 Integrating variant calling in multi-sequencing pipelines leveraging machine learning architectures
EP4602608A1 (en) 2022-10-11 2025-08-20 Illumina, Inc. Detecting and correcting methylation values from methylation sequencing assays
EP4482979B1 (en) 2022-11-09 2025-10-01 10X Genomics, Inc. Methods, compositions, and kits for determining the location of multiple analytes in a biological sample
EP4627583A1 (en) 2022-11-30 2025-10-08 Illumina, Inc. Accurately predicting variants from methylation sequencing data
WO2024129672A1 (en) 2022-12-12 2024-06-20 The Broad Institute, Inc. Trafficked rnas for assessment of cell-cell connectivity and neuroanatomy
EP4634400A1 (en) 2022-12-14 2025-10-22 Illumina, Inc. Systems and methods for capture and enrichment of clustered beads on flow cell substrates
CN119095982A (en) 2022-12-22 2024-12-06 伊路米纳有限公司 Palladium catalyst compositions and methods for sequencing by synthesis
WO2024137765A1 (en) 2022-12-22 2024-06-27 Illumina, Inc. Transition-metal catalyst compositions and methods for sequencing by synthesis
CN119095984A (en) 2022-12-27 2024-12-06 伊路米纳有限公司 Sequencing method using 3' allyl-terminated nucleotides
EP4655392A2 (en) 2023-01-27 2025-12-03 Universiteit Maastricht Methods for selecting cultured corneal endothelial cells
EP4662663A1 (en) 2023-02-06 2025-12-17 Illumina, Inc. Determining and removing inter-cluster light interference
KR20250169957A (en) 2023-03-30 2025-12-04 일루미나, 인코포레이티드 AI-driven signal enhancement of low-resolution images
EP4689181A1 (en) 2023-03-30 2026-02-11 Illumina, Inc. Compositions and methods for nucleic acid sequencing
EP4706045A1 (en) 2023-03-30 2026-03-11 Illumina, Inc. Tandem repeat genotyping
CA3260664A1 (en) 2023-05-03 2024-11-07 Illumina, Inc. Machine learning model for recalibrating genotype calls from existing sequencing data files
EP4720334A1 (en) 2023-05-26 2026-04-08 Illumina, Inc. Methods for preserving methylation status during clustering
EP4721076A1 (en) 2023-05-31 2026-04-08 Illumina, Inc. Improving structural variant alignment and variant calling by utilizing a structural-variant reference genome
WO2024249591A1 (en) 2023-05-31 2024-12-05 Illumina, Inc. Methods for double-stranded sequencing by synthesis
WO2024249973A2 (en) 2023-06-02 2024-12-05 Illumina, Inc. Linking human genes to clinical phenotypes using graph neural networks
WO2025006565A1 (en) 2023-06-27 2025-01-02 Illumina, Inc. Variant calling with methylation-level estimation
WO2025006874A1 (en) 2023-06-30 2025-01-02 Illumina, Inc. Machine-learning model for recalibrating genotype calls corresponding to germline variants and somatic mosaic variants
WO2025054389A1 (en) 2023-09-07 2025-03-13 Illumina, Inc. Identification of methylated cytosine using landmarks
WO2025058517A1 (en) 2023-09-12 2025-03-20 Levels Diagnostics Holding B.V. Biomarkers for typing a sample of an individual for hepatocellular carcinoma.
WO2025072783A1 (en) 2023-09-28 2025-04-03 Illumina, Inc. Altered cytidine deaminases and methods of use
US20250111899A1 (en) 2023-09-29 2025-04-03 Illumina, Inc. Predicting insert lengths using primary analysis metrics
WO2025072870A1 (en) 2023-09-29 2025-04-03 Illumina, Inc. Tracking and modifying cluster location on nucleotide-sample slides in real time
WO2025081064A2 (en) 2023-10-11 2025-04-17 Illumina, Inc. Thermophilic deaminase and methods for identifying modified cytosine
WO2025090883A1 (en) 2023-10-27 2025-05-01 Illumina, Inc. Detecting variants in nucleotide sequences based on haplotype diversity
WO2025117738A1 (en) 2023-11-28 2025-06-05 Illumina, Inc. Methods of improving unique molecular index ligation efficiency
US20250201342A1 (en) 2023-12-15 2025-06-19 Illumina, Inc. Minimal residual disease (mrd) models for determining likelihoods or probabilities of a subject comprising cancer
US20250201346A1 (en) 2023-12-18 2025-06-19 Illumina, Inc. Using machine learning models for detecting minimum residual disease (mrd) in a subject
WO2025137222A1 (en) 2023-12-19 2025-06-26 Illumina, Inc. Methylation detection assay
US20250210137A1 (en) 2023-12-20 2025-06-26 Illumina, Inc. Directly determining signal-to-noise-ratio metrics for accelerated convergence in determining nucleotide-base calls and base-call quality
WO2025136105A1 (en) 2023-12-20 2025-06-26 Stichting Amsterdam UMC Intestinal tissue-adherent microbial signatures predictive of response to anti-tnf-alpha in crohn's disease
US20250210141A1 (en) 2023-12-21 2025-06-26 Illumina, Inc. Enhanced mapping and alignment of nucleotide reads utilizing an improved haplotype data structure with allele-variant differences
WO2025144716A1 (en) 2023-12-28 2025-07-03 Illumina, Inc. Nucleotides with enzymatically cleavable 3'-o-glycoside blocking groups for sequencing
WO2025160089A1 (en) 2024-01-26 2025-07-31 Illumina, Inc. Custom multigenome reference construction for improved sequencing analysis of genomic samples
WO2025174774A1 (en) 2024-02-12 2025-08-21 Illumina, Inc. Determining offline corrections for sequence specific errors caused by low complexity nucleotide sequences
WO2025174708A1 (en) 2024-02-13 2025-08-21 Illumina, Inc. Design and method for cross-sequencing platform compatibility of flow cells
WO2025184226A1 (en) 2024-02-28 2025-09-04 Illumina, Inc. Nucleotides with terminal phosphate capping
WO2025184234A1 (en) 2024-02-28 2025-09-04 Illumina, Inc. A personalized haplotype database for improved mapping and alignment of nucleotide reads and improved genotype calling
WO2025188906A1 (en) 2024-03-08 2025-09-12 Illumina, Inc. Modified adenosine nucleotides
WO2025193747A1 (en) 2024-03-12 2025-09-18 Illumina, Inc. Machine-learning models for ordering and expediting sequencing tasks or corresponding nucleotide-sample slides
WO2025207535A1 (en) 2024-03-26 2025-10-02 Illumina, Inc. Photo-switchable surfaces
WO2025203031A2 (en) 2024-03-26 2025-10-02 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Methods of cancer detection by discordant methylation in cfdna
WO2025230914A1 (en) 2024-04-29 2025-11-06 Illumina, Inc. Nucleotides with enzyme-triggered self-immolative linkers for sequencing by synthesis
WO2025240241A1 (en) 2024-05-13 2025-11-20 Illumina, Inc. Modifying sequencing cycles during a sequencing run to meet customized coverage estimations for a target genomic region
WO2025240924A1 (en) 2024-05-17 2025-11-20 Illumina, Inc. Blind equalization systems for base calling applications
WO2025240905A1 (en) 2024-05-17 2025-11-20 The Broad Institute, Inc. Imaging-free high-resolution spatial macromolecule abundance reconstruction
WO2025250996A2 (en) 2024-05-31 2025-12-04 Illumina, Inc. Call generation and recalibration models for implementing personalized diploid reference haplotypes in genotype calling
WO2025264836A1 (en) 2024-06-18 2025-12-26 Illumina, Inc. Methods for increasing sequencing quality of gc-rich regions
WO2025264831A1 (en) 2024-06-18 2025-12-26 Illumina, Inc. Methods for increasing sequencing quality of gc-rich regions
WO2026006314A1 (en) 2024-06-28 2026-01-02 Illumina, Inc. Tagging target regions prior to nucleotide sequencing
WO2026006774A1 (en) 2024-06-28 2026-01-02 Illumina, Inc. Altered cytidine deaminases and methods of use
WO2026006771A1 (en) 2024-06-28 2026-01-02 Illumina, Inc. Cluster-filtering scores
WO2026030370A1 (en) 2024-07-30 2026-02-05 Illumina, Inc. Generating adaptive intensity value decision boundaries for nucleotide sequencing
WO2026072725A1 (en) 2024-09-25 2026-04-02 Illumina, Inc. Adjusting oligonucleotide-cluster location in images in real time using band-edge spectral regions
WO2026072986A1 (en) 2024-09-27 2026-04-02 Illumina, Inc. Determining offline corrections for sequence specific errors caused by low complexity sequences using a machine learning model

Family Cites Families (42)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5171534A (en) 1984-01-16 1992-12-15 California Institute Of Technology Automated DNA sequencing technique
US5821058A (en) 1984-01-16 1998-10-13 California Institute Of Technology Automated DNA sequencing technique
US4863849A (en) 1985-07-18 1989-09-05 New York Medical College Automatable process for sequencing nucleotide
US4811218A (en) 1986-06-02 1989-03-07 Applied Biosystems, Inc. Real time scanning electrophoresis apparatus for DNA sequencing
US5525464A (en) 1987-04-01 1996-06-11 Hyseq, Inc. Method of sequencing by hybridization of oligonucleotide probes
US4971903A (en) 1988-03-25 1990-11-20 Edward Hyman Pyrophosphate-based method and apparatus for sequencing nucleic acids
US5800992A (en) 1989-06-07 1998-09-01 Fodor; Stephen P.A. Method of detecting nucleic acids
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
US5302509A (en) 1989-08-14 1994-04-12 Beckman Instruments, Inc. Method for sequencing polynucleotides
US5445971A (en) * 1992-03-20 1995-08-29 Abbott Laboratories Magnetically assisted binding assays using magnetically labeled binding members
US5714320A (en) 1993-04-15 1998-02-03 University Of Rochester Rolling circle synthesis of oligonucleotides and amplification of select randomized circular oligonucleotides
US5482845A (en) 1993-09-24 1996-01-09 The Trustees Of Columbia University In The City Of New York Method for construction of normalized cDNA libraries
US5928905A (en) 1995-04-18 1999-07-27 Glaxo Group Limited End-complementary polymerase reaction
US5834252A (en) 1995-04-18 1998-11-10 Glaxo Group Limited End-complementary polymerase reaction
US5604097A (en) 1994-10-13 1997-02-18 Spectragen, Inc. Methods for sorting polynucleotides using oligonucleotide tags
US5919673A (en) * 1995-03-22 1999-07-06 The Scripps Research Institute One-pot enzymatic sulfation process using 3'-phosphoadenosine-5'-phosphosulfate and recycled phosphorylated adenosine intermediates
US5648245A (en) 1995-05-09 1997-07-15 Carnegie Institution Of Washington Method for constructing an oligonucleotide concatamer library by rolling circle replication
US5728529A (en) 1995-06-23 1998-03-17 Baylor College Of Medicine Alternative dye-labeled ribonucleotides, deoxyribonucleotides, and dideoxyribonucleotides for automated DNA analysis
US5871697A (en) 1995-10-24 1999-02-16 Curagen Corporation Method and apparatus for identifying, classifying, or quantifying DNA sequences in a sample without sequencing
DK0862656T3 (en) * 1995-11-21 2001-04-09 Univ Yale Unimolecular segment amplification and detection
US5854033A (en) 1995-11-21 1998-12-29 Yale University Rolling circle replication reporter systems
US5837196A (en) * 1996-01-26 1998-11-17 The Regents Of The University Of California High density array fabrication and readout method for a fiber optic biosensor
US5851772A (en) 1996-01-29 1998-12-22 University Of Chicago Microchip method for the enrichment of specific DNA sequences
US5712127A (en) 1996-04-29 1998-01-27 Genescape Inc. Subtractive amplification
US5846727A (en) 1996-06-06 1998-12-08 Board Of Supervisors Of Louisiana State University And Agricultural & Mechanical College Microsystem for rapid DNA sequencing
GB9618050D0 (en) 1996-08-29 1996-10-09 Cancer Res Campaign Tech Global amplification of nucleic acids
US5846721A (en) 1996-09-19 1998-12-08 The Trustees Of Columbia University In The City Of New York Efficient and simpler method to construct normalized cDNA libraries with improved representations of full-length cDNAs
GB9620209D0 (en) 1996-09-27 1996-11-13 Cemu Bioteknik Ab Method of sequencing DNA
WO1998020019A1 (en) * 1996-11-06 1998-05-14 Sequenom, Inc. Compositions and methods for immobilizing nucleic acids to solid supports
WO1998035012A2 (en) * 1997-02-12 1998-08-13 Chan Eugene Y Methods and products for analyzing polymers
JP2001517948A (en) 1997-04-01 2001-10-09 グラクソ、グループ、リミテッド Nucleic acid sequencing
JP2002503954A (en) 1997-04-01 2002-02-05 グラクソ、グループ、リミテッド Nucleic acid amplification method
US6406845B1 (en) * 1997-05-05 2002-06-18 Trustees Of Tuft College Fiber optic biosensor for selectively detecting oligonucleotide species in a mixed fluid sample
JP4294740B2 (en) 1997-05-23 2009-07-15 ソレクサ・インコーポレイテッド System and apparatus for serial processing of analytes
JP2001512700A (en) 1997-08-07 2001-08-28 キュラジェン コーポレイション Detection and confirmation of nucleic acid sequences using oligonucleotides containing subsequences that hybridize strictly to known terminal sequences and subsequences that hybridize to unidentified sequences
US6399334B1 (en) 1997-09-24 2002-06-04 Invitrogen Corporation Normalized nucleic acid libraries and methods of production thereof
AU1623899A (en) 1997-12-04 1999-06-16 Packard Bioscience Company Methods of using probes for analyzing polynucleotide sequence
NL1007781C2 (en) 1997-12-12 1999-06-15 Packard Instr Bv Microtiter plate.
WO1999036576A1 (en) 1998-01-20 1999-07-22 Packard Bioscience Company Gel pad arrays and methods and systems for making them
US5882874A (en) 1998-02-27 1999-03-16 The Trustees Of Columbia University In The City Of New York Reciprocal subtraction differential display
WO1999053102A1 (en) 1998-04-16 1999-10-21 Packard Bioscience Company Analysis of polynucleotide sequence
WO2000063437A2 (en) * 1999-04-20 2000-10-26 Illumina, Inc. Detection of nucleic acid reactions on bead arrays

Cited By (192)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040191816A1 (en) * 1992-08-04 2004-09-30 Replicon, Inc. In vitro amplification of nucleic acid molecules via circular replicons
US20080305535A1 (en) * 1992-08-04 2008-12-11 Replicon, Inc. In vitro amplification of nucleic acid molecules via circular replicons
US7615625B2 (en) 1992-08-04 2009-11-10 Replicon, Inc. In vitro amplification of nucleic acid molecules via circular replicons
US9109251B2 (en) 2004-06-25 2015-08-18 University Of Hawaii Ultrasensitive biosensors
US10563252B2 (en) 2004-06-25 2020-02-18 University Of Hawaii Ultrasensitive biosensors
US9228971B2 (en) 2004-11-12 2016-01-05 The Board Of Trustees Of The Leland Stanford Junior University Charge perturbation detection system for DNA and other molecules
US10822641B2 (en) 2004-11-12 2020-11-03 The Board Of Trustees Of The Leland Stanford Junior University Charge perturbation detection system for DNA and other molecules
US8012756B2 (en) 2004-11-12 2011-09-06 The Board Of Trustees Of The Leland Stanford Junior University Charge perturbation detection system for DNA and other molecules
US12139749B2 (en) 2004-11-12 2024-11-12 The Board Of Trustees Of The Leland Stanford Junior University Charge perturbation detection system for DNA and other molecules
US7785785B2 (en) 2004-11-12 2010-08-31 The Board Of Trustees Of The Leland Stanford Junior University Charge perturbation detection system for DNA and other molecules
US20060105373A1 (en) * 2004-11-12 2006-05-18 The Board Of Trustees Of The Leland Stanford Junior University Charge perturbation detection system for DNA and other molecules
US8753812B2 (en) 2004-11-12 2014-06-17 The Board Of Trustees Of The Leland Stanford Junior University Charge perturbation detection method for DNA and other molecules
US8313907B2 (en) 2004-11-12 2012-11-20 The Board Of Trustees Of The Leland Stanford Junior University Charge perturbation detection system for DNA and other molecules
US11378498B2 (en) 2006-06-14 2022-07-05 Verinata Health, Inc. Diagnosis of fetal abnormalities using polymorphisms including short tandem repeats
US11781187B2 (en) * 2006-06-14 2023-10-10 The General Hospital Corporation Rare cell analysis using sample splitting and DNA tags
US8766328B2 (en) 2006-12-14 2014-07-01 Life Technologies Corporation Chemically-sensitive sample and hold sensors
US7948015B2 (en) 2006-12-14 2011-05-24 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US10633699B2 (en) 2006-12-14 2020-04-28 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US20090026082A1 (en) * 2006-12-14 2009-01-29 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes using large scale FET arrays
US8264014B2 (en) 2006-12-14 2012-09-11 Life Technologies Corporation 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
US10502708B2 (en) 2006-12-14 2019-12-10 Life Technologies Corporation Chemically-sensitive sensor array calibration circuitry
US8269261B2 (en) 2006-12-14 2012-09-18 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US8293082B2 (en) 2006-12-14 2012-10-23 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US20090127589A1 (en) * 2006-12-14 2009-05-21 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes using large scale FET arrays
US8313639B2 (en) 2006-12-14 2012-11-20 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US8313625B2 (en) 2006-12-14 2012-11-20 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US8317999B2 (en) 2006-12-14 2012-11-27 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US8349167B2 (en) 2006-12-14 2013-01-08 Life Technologies Corporation Methods and apparatus for detecting molecular interactions using FET arrays
US10415079B2 (en) 2006-12-14 2019-09-17 Life Technologies Corporation Methods and apparatus for detecting molecular interactions using FET arrays
US11435314B2 (en) 2006-12-14 2022-09-06 Life Technologies Corporation Chemically-sensitive sensor array device
US8415716B2 (en) 2006-12-14 2013-04-09 Life Technologies Corporation Chemically sensitive sensors with feedback circuits
US10203300B2 (en) 2006-12-14 2019-02-12 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US8426899B2 (en) 2006-12-14 2013-04-23 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US8426898B2 (en) 2006-12-14 2013-04-23 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US20220340965A1 (en) * 2006-12-14 2022-10-27 Life Technologies Corporation Methods and Apparatus for Measuring Analytes Using Large Scale FET Arrays
US11732297B2 (en) * 2006-12-14 2023-08-22 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US8435395B2 (en) 2006-12-14 2013-05-07 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US8441044B2 (en) 2006-12-14 2013-05-14 Life Technologies Corporation Methods for manufacturing low noise chemically-sensitive field effect transistors
US8445945B2 (en) 2006-12-14 2013-05-21 Life Technologies Corporation Low noise chemically-sensitive field effect transistors
US9989489B2 (en) 2006-12-14 2018-06-05 Life Technnologies Corporation Methods for calibrating an array of chemically-sensitive sensors
US9951382B2 (en) 2006-12-14 2018-04-24 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US9404920B2 (en) 2006-12-14 2016-08-02 Life Technologies Corporation Methods and apparatus for detecting molecular interactions using FET arrays
US9269708B2 (en) 2006-12-14 2016-02-23 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US8492800B2 (en) 2006-12-14 2013-07-23 Life Technologies Corporation Chemically sensitive sensors with sample and hold capacitors
US8492799B2 (en) 2006-12-14 2013-07-23 Life Technologies Corporation Methods and apparatus for detecting molecular interactions using FET arrays
US8496802B2 (en) 2006-12-14 2013-07-30 Life Technologies Corporation Methods for operating chemically-sensitive sample and hold sensors
US8502278B2 (en) 2006-12-14 2013-08-06 Life Technologies Corporation Chemically-sensitive sample and hold sensors
US8519448B2 (en) 2006-12-14 2013-08-27 Life Technologies Corporation Chemically-sensitive array with active and reference sensors
US20100188073A1 (en) * 2006-12-14 2010-07-29 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes using large scale fet arrays
US9134269B2 (en) 2006-12-14 2015-09-15 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US8530941B2 (en) 2006-12-14 2013-09-10 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US20110230375A1 (en) * 2006-12-14 2011-09-22 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale fet arrays
US8535513B2 (en) 2006-12-14 2013-09-17 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US8540868B2 (en) 2006-12-14 2013-09-24 Life Technologies Corporation Methods and apparatus for detecting molecular interactions using FET arrays
US8540866B2 (en) 2006-12-14 2013-09-24 Life Technologies Corporation Methods and apparatus for detecting molecular interactions using FET arrays
US8540865B2 (en) 2006-12-14 2013-09-24 Life Technologies Corporation Methods and apparatus for detecting molecular interactions using FET arrays
US8540867B2 (en) 2006-12-14 2013-09-24 Life Technologies Corporation Methods and apparatus for detecting molecular interactions using FET arrays
US12066399B2 (en) 2006-12-14 2024-08-20 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US8558288B2 (en) 2006-12-14 2013-10-15 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US8575664B2 (en) 2006-12-14 2013-11-05 Life Technologies Corporation Chemically-sensitive sensor array calibration circuitry
US20100197507A1 (en) * 2006-12-14 2010-08-05 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes using large scale fet arrays
US9039888B2 (en) 2006-12-14 2015-05-26 Life Technologies Corporation Methods and apparatus for detecting molecular interactions using FET arrays
US9023189B2 (en) 2006-12-14 2015-05-05 Life Technologies Corporation High density sensor array without wells
US8658017B2 (en) 2006-12-14 2014-02-25 Life Technologies Corporation Methods for operating an array of chemically-sensitive sensors
US12140560B2 (en) 2006-12-14 2024-11-12 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US8685230B2 (en) 2006-12-14 2014-04-01 Life Technologies Corporation Methods and apparatus for high-speed operation of a chemically-sensitive sensor array
US8890216B2 (en) 2006-12-14 2014-11-18 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US8692298B2 (en) 2006-12-14 2014-04-08 Life Technologies Corporation Chemical sensor array having multiple sensors per well
US8450781B2 (en) 2006-12-14 2013-05-28 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US8306757B2 (en) 2006-12-14 2012-11-06 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US8764969B2 (en) 2006-12-14 2014-07-01 Life Technologies Corporation Methods for operating chemically sensitive sensors with sample and hold capacitors
US8742472B2 (en) 2006-12-14 2014-06-03 Life Technologies Corporation Chemically sensitive sensors with sample and hold capacitors
US10816506B2 (en) 2006-12-14 2020-10-27 Life Technologies Corporation Method for measuring analytes using large scale chemfet arrays
US8551704B2 (en) 2007-02-16 2013-10-08 Pacific Biosciences Of California, Inc. Controllable strand scission of mini circle DNA
US11339430B2 (en) 2007-07-10 2022-05-24 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US8535882B2 (en) 2007-07-26 2013-09-17 Pacific Biosciences Of California, Inc. Molecular redundant sequencing
US9732383B2 (en) 2007-07-26 2017-08-15 Pacific Biosciences Of California, Inc. Molecular redundant sequencing
US20090029385A1 (en) * 2007-07-26 2009-01-29 Pacific Biosciences Of California, Inc. Molecular redundant sequencing
US9051611B2 (en) 2007-07-26 2015-06-09 Pacific Biosciences Of California, Inc. Molecular redundant sequencing
WO2009017678A3 (en) * 2007-07-26 2009-04-16 Pacific Biosciences California Molecular redundant sequencing
US20110212436A1 (en) * 2007-07-26 2011-09-01 Pacific Biosciences Of California, Inc. Molecular redundant sequencing
US7901889B2 (en) 2007-07-26 2011-03-08 Pacific Biosciences Of California, Inc. Molecular redundant sequencing
US20090270273A1 (en) * 2008-04-21 2009-10-29 Complete Genomics, Inc. Array structures for nucleic acid detection
US8470164B2 (en) 2008-06-25 2013-06-25 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US8524057B2 (en) 2008-06-25 2013-09-03 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US9194000B2 (en) 2008-06-25 2015-11-24 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US9944981B2 (en) 2008-10-22 2018-04-17 Life Technologies Corporation Methods and apparatus for measuring analytes
US8936763B2 (en) 2008-10-22 2015-01-20 Life Technologies Corporation Integrated sensor arrays for biological and chemical analysis
US11874250B2 (en) 2008-10-22 2024-01-16 Life Technologies Corporation Integrated sensor arrays for biological and chemical analysis
US9964515B2 (en) 2008-10-22 2018-05-08 Life Technologies Corporation Integrated sensor arrays for biological and chemical analysis
US11448613B2 (en) 2008-10-22 2022-09-20 Life Technologies Corporation ChemFET sensor array including overlying array of wells
US11137369B2 (en) 2008-10-22 2021-10-05 Life Technologies Corporation Integrated sensor arrays for biological and chemical analysis
US12146853B2 (en) 2008-10-22 2024-11-19 Life Technologies Corporation Methods and apparatus including array of reaction chambers over array of chemFET sensors for measuring analytes
US11692964B2 (en) 2009-05-29 2023-07-04 Life Technologies Corporation Methods and apparatus for measuring analytes
US12038405B2 (en) 2009-05-29 2024-07-16 Life Technologies Corporation Methods and apparatus for measuring analytes
US10809226B2 (en) 2009-05-29 2020-10-20 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
US8748947B2 (en) 2009-05-29 2014-06-10 Life Technologies Corporation Active chemically-sensitive sensors with reset switch
US8994076B2 (en) 2009-05-29 2015-03-31 Life Technologies Corporation Chemically-sensitive field effect transistor based pixel array with protection diodes
US10718733B2 (en) 2009-05-29 2020-07-21 Life Technologies Corporation Methods and apparatus for measuring analytes
US8592154B2 (en) 2009-05-29 2013-11-26 Life Technologies Corporation Methods and apparatus for high speed operation of a chemically-sensitive sensor array
US8742469B2 (en) 2009-05-29 2014-06-03 Life Technologies Corporation Active chemically-sensitive sensors with correlated double sampling
US8263336B2 (en) 2009-05-29 2012-09-11 Life Technologies Corporation Methods and apparatus for measuring analytes
US10451585B2 (en) 2009-05-29 2019-10-22 Life Technologies Corporation Methods and apparatus for measuring analytes
US8766327B2 (en) 2009-05-29 2014-07-01 Life Technologies Corporation Active chemically-sensitive sensors with in-sensor current sources
US8592153B1 (en) 2009-05-29 2013-11-26 Life Technologies Corporation Methods for manufacturing high capacitance microwell structures of chemically-sensitive sensors
US8822205B2 (en) 2009-05-29 2014-09-02 Life Technologies Corporation Active chemically-sensitive sensors with source follower amplifier
US8912580B2 (en) 2009-05-29 2014-12-16 Life Technologies Corporation Active chemically-sensitive sensors with in-sensor current sources
US8776573B2 (en) 2009-05-29 2014-07-15 Life Technologies Corporation Methods and apparatus for measuring analytes
US12461054B2 (en) 2009-05-29 2025-11-04 Life Technologies Corporation Apparatus and methods for performing electrochemical reactions
US8698212B2 (en) 2009-05-29 2014-04-15 Life Technologies Corporation Active chemically-sensitive sensors
US9927393B2 (en) 2009-05-29 2018-03-27 Life Technologies Corporation Methods and apparatus for measuring analytes
US11768171B2 (en) 2009-05-29 2023-09-26 Life Technologies Corporation Methods and apparatus for measuring analytes
US8455927B2 (en) 2010-06-30 2013-06-04 Life Technologies Corporation One-transistor pixel array with cascoded column circuit
US8432150B2 (en) 2010-06-30 2013-04-30 Life Technologies Corporation Methods for operating an array column integrator
US8983783B2 (en) 2010-06-30 2015-03-17 Life Technologies Corporation Chemical detection device having multiple flow channels
US12038406B2 (en) 2010-06-30 2024-07-16 Life Technologies Corporation Semiconductor-based chemical detection device
US10641729B2 (en) 2010-06-30 2020-05-05 Life Technologies Corporation Column ADC
US8487790B2 (en) 2010-06-30 2013-07-16 Life Technologies Corporation Chemical detection circuit including a serializer circuit
US8217433B1 (en) 2010-06-30 2012-07-10 Life Technologies Corporation One-transistor pixel array
US11231451B2 (en) 2010-06-30 2022-01-25 Life Technologies Corporation Methods and apparatus for testing ISFET arrays
US8247849B2 (en) 2010-06-30 2012-08-21 Life Technologies Corporation Two-transistor pixel array
US10481123B2 (en) 2010-06-30 2019-11-19 Life Technologies Corporation Ion-sensing charge-accumulation circuits and methods
US8823380B2 (en) 2010-06-30 2014-09-02 Life Technologies Corporation Capacitive charge pump
US9239313B2 (en) 2010-06-30 2016-01-19 Life Technologies Corporation Ion-sensing charge-accumulation circuits and methods
US9164070B2 (en) 2010-06-30 2015-10-20 Life Technologies Corporation Column adc
US8524487B2 (en) 2010-06-30 2013-09-03 Life Technologies Corporation One-transistor pixel array with cascoded column circuit
US8858782B2 (en) 2010-06-30 2014-10-14 Life Technologies Corporation Ion-sensing charge-accumulation circuits and methods
US8741680B2 (en) 2010-06-30 2014-06-03 Life Technologies Corporation Two-transistor pixel array
US8415177B2 (en) 2010-06-30 2013-04-09 Life Technologies Corporation Two-transistor pixel array
US8772698B2 (en) 2010-06-30 2014-07-08 Life Technologies Corporation CCD-based multi-transistor active pixel sensor array
US8415176B2 (en) 2010-06-30 2013-04-09 Life Technologies Corporation One-transistor pixel array
US8421437B2 (en) 2010-06-30 2013-04-16 Life Technologies Corporation Array column integrator
US8742471B2 (en) 2010-06-30 2014-06-03 Life Technologies Corporation Chemical sensor array with leakage compensation circuit
US8432149B2 (en) 2010-06-30 2013-04-30 Life Technologies Corporation Array column integrator
US8731847B2 (en) 2010-06-30 2014-05-20 Life Technologies Corporation Array configuration and readout scheme
US11307166B2 (en) 2010-07-01 2022-04-19 Life Technologies Corporation Column ADC
US9960253B2 (en) 2010-07-03 2018-05-01 Life Technologies Corporation Chemically sensitive sensor with lightly doped drains
US8653567B2 (en) 2010-07-03 2014-02-18 Life Technologies Corporation Chemically sensitive sensor with lightly doped drains
US9618475B2 (en) 2010-09-15 2017-04-11 Life Technologies Corporation Methods and apparatus for measuring analytes
US12050195B2 (en) 2010-09-15 2024-07-30 Life Technologies Corporation Methods and apparatus for measuring analytes using chemfet arrays
US9958414B2 (en) 2010-09-15 2018-05-01 Life Technologies Corporation Apparatus for measuring analytes including chemical sensor array
US9958415B2 (en) 2010-09-15 2018-05-01 Life Technologies Corporation ChemFET sensor including floating gate
US8685324B2 (en) 2010-09-24 2014-04-01 Life Technologies Corporation Matched pair transistor circuits
US9110015B2 (en) 2010-09-24 2015-08-18 Life Technologies Corporation Method and system for delta double sampling
US8796036B2 (en) 2010-09-24 2014-08-05 Life Technologies Corporation Method and system for delta double sampling
US8912005B1 (en) 2010-09-24 2014-12-16 Life Technologies Corporation Method and system for delta double sampling
US9970984B2 (en) 2011-12-01 2018-05-15 Life Technologies Corporation Method and apparatus for identifying defects in a chemical sensor array
US10365321B2 (en) 2011-12-01 2019-07-30 Life Technologies Corporation Method and apparatus for identifying defects in a chemical sensor array
US10598723B2 (en) 2011-12-01 2020-03-24 Life Technologies Corporation Method and apparatus for identifying defects in a chemical sensor array
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
US10404249B2 (en) 2012-05-29 2019-09-03 Life Technologies Corporation System for reducing noise in a chemical sensor array
US8786331B2 (en) 2012-05-29 2014-07-22 Life Technologies Corporation System for reducing noise in a chemical sensor array
US9270264B2 (en) 2012-05-29 2016-02-23 Life Technologies Corporation System for reducing noise in a chemical sensor array
US9985624B2 (en) 2012-05-29 2018-05-29 Life Technologies Corporation System for reducing noise in a chemical sensor array
US8552771B1 (en) 2012-05-29 2013-10-08 Life Technologies Corporation System for reducing noise in a chemical sensor array
US9080968B2 (en) 2013-01-04 2015-07-14 Life Technologies Corporation Methods and systems for point of use removal of sacrificial material
US9852919B2 (en) 2013-01-04 2017-12-26 Life Technologies Corporation Methods and systems for point of use removal of sacrificial material
US10436742B2 (en) 2013-01-08 2019-10-08 Life Technologies Corporation Methods for manufacturing well structures for low-noise chemical sensors
US9841398B2 (en) 2013-01-08 2017-12-12 Life Technologies Corporation Methods for manufacturing well structures for low-noise chemical sensors
US8962366B2 (en) 2013-01-28 2015-02-24 Life Technologies Corporation Self-aligned well structures for low-noise chemical sensors
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
US9995708B2 (en) 2013-03-13 2018-06-12 Life Technologies Corporation Chemical sensor with sidewall spacer sensor surface
US9116117B2 (en) 2013-03-15 2015-08-25 Life Technologies Corporation Chemical sensor with sidewall sensor surface
US9128044B2 (en) 2013-03-15 2015-09-08 Life Technologies Corporation Chemical sensors with consistent sensor surface areas
US10481124B2 (en) 2013-03-15 2019-11-19 Life Technologies Corporation Chemical device with thin conductive element
US9671363B2 (en) 2013-03-15 2017-06-06 Life Technologies Corporation Chemical sensor with consistent sensor surface areas
US10422767B2 (en) 2013-03-15 2019-09-24 Life Technologies Corporation Chemical sensor with consistent sensor surface areas
US9823217B2 (en) 2013-03-15 2017-11-21 Life Technologies Corporation Chemical device with thin conductive element
US9835585B2 (en) 2013-03-15 2017-12-05 Life Technologies Corporation Chemical sensor with protruded sensor surface
US10655175B2 (en) 2013-05-09 2020-05-19 Life Technologies Corporation Windowed sequencing
US10100357B2 (en) 2013-05-09 2018-10-16 Life Technologies Corporation Windowed sequencing
US11028438B2 (en) 2013-05-09 2021-06-08 Life Technologies Corporation Windowed sequencing
US11499938B2 (en) 2013-06-10 2022-11-15 Life Technologies Corporation Chemical sensor array having multiple sensors per well
US10458942B2 (en) 2013-06-10 2019-10-29 Life Technologies Corporation Chemical sensor array having multiple sensors per well
US11774401B2 (en) 2013-06-10 2023-10-03 Life Technologies Corporation Chemical sensor array having multiple sensors per well
US10816504B2 (en) 2013-06-10 2020-10-27 Life Technologies Corporation Chemical sensor array having multiple sensors per well
US11536688B2 (en) 2014-12-18 2022-12-27 Life Technologies Corporation High data rate integrated circuit with transmitter configuration
US10077472B2 (en) 2014-12-18 2018-09-18 Life Technologies Corporation High data rate integrated circuit with power management
US10379079B2 (en) 2014-12-18 2019-08-13 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US10605767B2 (en) 2014-12-18 2020-03-31 Life Technologies Corporation High data rate integrated circuit with transmitter configuration
US12196704B2 (en) 2014-12-18 2025-01-14 Life Technologies Corporation High data rate integrated circuit with transmitter configuration
US10767224B2 (en) 2014-12-18 2020-09-08 Life Technologies Corporation High data rate integrated circuit with power management
WO2016170182A1 (en) 2015-04-24 2016-10-27 Qiagen Gmbh Method for immobilizing a nucleic acid molecule on a solid support
WO2016170179A1 (en) 2015-04-24 2016-10-27 Qiagen Gmbh Method for immobilizing a nucleic acid molecule on solid support
US11220705B2 (en) 2015-04-24 2022-01-11 Qiagen Gmbh Method for immobilizing a nucleic acid molecule on solid support
US11085073B2 (en) 2015-04-24 2021-08-10 Qiagen Gmbh Method for immobilizing a nucleic acid molecule on a solid support
WO2018137826A1 (en) 2017-01-26 2018-08-02 Qiagen Gmbh Method for enriching template nucleic acids
EP3929306A1 (en) 2017-01-26 2021-12-29 QIAGEN GmbH Method for enriching template nucleic acids

Also Published As

Publication number Publication date
WO2001020039A3 (en) 2002-03-21
AU784708B2 (en) 2006-06-01
CA2384510C (en) 2011-11-15
JP2003514514A (en) 2003-04-22
AU7582000A (en) 2001-04-17
JP4727109B2 (en) 2011-07-20
CA2384510A1 (en) 2001-03-22
US6274320B1 (en) 2001-08-14
EP1212467A2 (en) 2002-06-12
WO2001020039A2 (en) 2001-03-22

Similar Documents

Publication Publication Date Title
US6274320B1 (en) Method of sequencing a nucleic acid
US7244559B2 (en) Method of sequencing a nucleic acid
US7211390B2 (en) Method of sequencing a nucleic acid
CA2513899C (en) Methods of amplifying and sequencing nucleic acids
US7575865B2 (en) Methods of amplifying and sequencing nucleic acids
US20030054396A1 (en) Enzymatic light amplification
WO2003087388A9 (en) Bioluminescence regenerative cycle (brc) for nucleic acid quantification

Legal Events

Date Code Title Description
STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION