System and methods for nucleic acid sequencing of single molecules by polymerase synthesis

{
  "publication_number": "US8192961B2",
  "country": "US",
  "kind": "B2",
  "title": "System and methods for nucleic acid sequencing of single molecules by polymerase synthesis",
  "abstract": "This invention relates to improved methods for sequencing and genotyping nucleic acid in a single molecule configuration. The method involves single molecule detection of fluorescent labeled PPi moieties released from NTPs as a polymerase extension product is created.",
  "claims": [
    "1. A method for identifying a nucleotide in a template nucleic acid sequence, said method comprising: a) providing a complex of a polymerase, a template nucleic acid sequence and a primer sequence, wherein said polymerase or said template nucleic acid sequence is immobilized on a solid support, b) contacting said complex with one or more types of optically labeled nucleotides, wherein the one of said one or more types of optically labeled nucleotides are unblocked at the 3′OH and each of the one or more types of optically labeled nucleotides comprises a base and a γ-phosphate having an optical label; c) producing an extended primer by incorporation of the bases of the one or more optically labeled nucleotides in a primer extension reaction and detecting said incorporation by cleaving said γ-phosphate from each of one or more nucleotides whose base or bases is/are incorporated into said extended primer and detecting an optical signal produced by said optical label on said γ-phosphate; and d) identifying the nucleotide in said template nucleic acid sequence.",
    "2. The method of claim 1, wherein the contacting step comprises contacting the complex with more than one nucleotide of the one or more types of optically labeled nucleotides at a time.",
    "3. The method of claim 1, wherein the cleaved γ-phosphate is washed away.",
    "4. The method of claim 1, wherein said optical label is a fluorescent label.",
    "5. The method of claim 4, wherein said detecting an optical signal produced by said optical label is carried out using a total internal reflection fluorescence microscope.",
    "6. The method of claim 1, further comprising washing the optically labeled nucleotides unincorporated to said extended primer away from said complex.",
    "7. The method of claim 1, wherein said template nucleic acid comprises a single nucleotide polymorphism.",
    "8. The method of claim 1, wherein said method comprises detecting, a unique optical signal from the one or more optically labeled nucleotides using a system or device selected from the group that consists of a high-efficiency photon detection system, a photodiode, a camera, a charge couple device, an intensified charge couple device, a near-field scanning microscope, a far-field confocal microscope, and a microscope that detects wide-field epi-illumination.",
    "9. The method of claim 8, wherein said detecting step is carried out in an evanescent optical field.",
    "10. The method of claim 1, wherein the contacting step comprises contacting the complex with more than one type of said optically labeled nucleotides, and the detection is a sequential detection of said optical signal produced by said optical label on said γ-phosphate from said each of one or more nucleotides whose base or bases is/are incorporated into said extended primer.",
    "11. The method of claim 1, wherein said method further comprises contacting a solution with said solid support and flowing said solution past the immobilized polymerase or template nucleic acid.",
    "12. The method of claim 1, wherein said detecting step further comprises acquiring a sequence of images.",
    "13. The method of claim 12, wherein said sequence of images is analyzed to track single molecules.",
    "14. The method of claim 1, wherein said detecting step is carried out by a mechanism that provides low background and enables the detection of single molecules.",
    "15. The method of claim 1, wherein said solid support comprises an array of template nucleic acids or polymerases.",
    "16. The method of claim 15, wherein said complex is immobilized through the template nucleic acid or the polymerase of said complex, wherein the template nucleic acid or the polymerase is in single molecules configuration.",
    "17. The method of claim 15, wherein the array comprises an array of locations on the solid support, each of said array of locations bearing a single molecule of polymerase or target nucleic acid.",
    "18. The method of claim 17, wherein each member of said array is located in a defined location.",
    "19. The method of claim 17, wherein each member of said array is different.",
    "20. The method of claim 17, wherein said array is addressable."
  ],
  "description_excerpt": "[Not Applicable]\n\nThis invention relates to improved methods for sequencing and genotyping nucleic acid in a single molecule configuration. The method involves single molecule detection of fluorescent labeled pyrophosphate [PPi] moieties released from nucleotide triphosphate [NTPs] as a polymerase extension product is created.\n\nPrevious non-electrophoretic sequencing and genotyping methods have not taken advantage of the continuous detection of PPi release from incorporated nucleotides. In the prior art of PPi sequencing (www.pyrosequencing.com), each nucleotide Adenosine-5′phosphate “A”, Cytidine 5′-phosphate “C”, Guanosine 5′-phosphate, Uridine 5′-phosphate “U” and Thymidine 5′-phosphate “T”, is added separately to a reaction mixture containing the nucleic acid target and a polymerase. The current nucleotide is removed before the next is added. Incorporation of a nucleotide is accompanied by release of PPi from the NTP, detected by a secondary assay for PPi. A secondary assay is required because the PPi moiety of the NTP is not labeled. Only one nucleotide can be tested per cycle; if all 4 NTPs were present simultaneously, the polymerization reaction would continue uncontrolled and no sequence information could be obtained. Read length is limited by loss of synchronization among the target nucleic acid molecules in the sample.\n\nOther non-electrophoretic methods, such as the stepwise ligation and cleavage of probes on DNA fragments attached to microbeads, requires synchronization of the DNA molecules that inevitably decays with each cycle.",
  "cpc": [
    "C07D 209/12",
    "C07H 19/10",
    "C07H 19/20",
    "C07H 21/00",
    "C12Q 1/6869",
    "C12Q 1/6874",
    "Y10T 436/143333"
  ],
  "ipc": [
    "C40B 20/04",
    "C07D 209/12",
    "C07H 19/10",
    "C07H 19/20",
    "C07H 21/00",
    "C12M 1/00",
    "C12N 11/14",
    "C12N 15/09",
    "C12Q 1/68",
    "C07H 21/02",
    "C07H 21/04",
    "C12M 1/34",
    "C12P 19/34"
  ],
  "assignees": [
    "Pacific Biosciences of California Inc"
  ],
  "inventors": [
    "John G. K. Williams"
  ],
  "filing_date": "2007-10-24",
  "publication_date": "2012-06-05",
  "grant_date": "2012-06-05",
  "priority_date": "1998-12-14",
  "application_number": "US-92359907-A",
  "family_id": "26809573",
  "cited_by_count": 93,
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