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

• US 8,192,961 B2
• Filed: 10/24/2007
• Issued: 06/05/2012
• Est. Priority Date: 12/14/1998
• Status: Expired due to Term

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Williams, John G. K.
• Primary Examiner(s): Lu, Frank W
• Application Number:
  US11/923,599
• Publication Number:
  US 20090082212A1
• Time in Patent Office: 1,686 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/91.51, 435/183, 435/6.1, 435/6.11, 435/6.12, 435/283.1, 435/287.1, 435/287.2, 436/94, 536/23.1, 536/24.3, 536/24.33, 536/25.3, 536/25.32
• US Class Current: 435/91.2
• CPC Class Codes: C07D 209/12 Radicals substituted by oxygen atoms
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

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.