System and method for nucleic acid sequencing by polymerase synthesis

• US 7,229,799 B2
• Filed: 05/14/2003
• Issued: 06/12/2007
• Est. Priority Date: 12/14/1998
• Status: Expired due to Term

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Li & S Company Limited (Yesco Holdings Co., Ltd.)
• Inventors: Williams, John G. K.
• Primary Examiner(s): LU, FRANK WEI MIN
• Application Number:
  US10/438,639
• Publication Number:
  US 20030194740A1
• Time in Patent Office: 1,490 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/91.51, 435/283.1, 435/287.1, 435/287.2, 435/183, 436/94, 536/23.1, 536/24.3, 536/24.33, 536/25.3, 536/25.32, 422/68.1
• 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 genotyping or sequencing a single target nucleic acid molecule, said method comprising:

i) immobilizing onto a solid support a target nucleic acid or a nucleic acid polymerase to form a solid support comprising more than one site or location each bearing only one single individual molecule of polymerase or target nucleic acid;

ii) contacting the solid support with the target nucleic acid when the polymerase is immobilized or the polymerase when the target nucleic acid is immobilized, a primer nucleic acid which complements a region of the target nucleic acid, at least two types of fluorescently labeled nucleotide triphosphates (NTPs), wherein each type of NTP has a different fluorescently label on the γ

-phosphate; and

iii) determining the incorporation of a fluorescently labeled NTP into a single molecule of the primer stand, by detecting the fluorescently labeled γ

-phosphate of the labeled NTP, which is then cleaved away from the fluorescently labeled NTP, thereby genotyping or sequencing said single target nucleic acid.

2. The method of claim 1, wherein the determination is further by a sequential incorporation of different types of fluorescently labeled NTP.

3. The method of claim 1, wherein the fluorescently labeled NTP is further labeled with a quencher moiety.

4. The method of claim 1, wherein a solution contacting the solid support flows past the immobilized polymerase or target nucleic acid.

5. The method claim 1, wherein the target nucleic acid is deoxynucleotide acid, the polymerase is a DNA dependent DNA polymerase and the fluorescently labeled NTP is a dNTP.

6. The method of claim 1, wherein the target nuclei acid is double stranded DNA and the polymerase is RNA polymerase and the fluorescently labeled NTP is a rNTP.

7. The method of claim 1, wherein said detecting is carried out using a system or device selected from the group consisting of an optical reader, 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, a microscope that detects wide-field epi-illumination, and a total internal reflection fluorescence microscope.

8. The method of claim 7, wherein said detecting comprises acquiring a sequence of images.

9. The method of claim 8, wherein the sequence of images is analyzed to track single individual molecules.

10. The method of claim 1, wherein said detecting is carried out by a mechanism that provides low background and enables the detection of single individual molecules.

11. The method of claim 7, wherein said detecting is carried out by an evanescent optical field.

12. The method of claim 7, wherein said detecting is carried out by a mechanism selected from the group consisting of flourescence resonance energy transfer, an electron transfer mechanism, an excited-state lifetime mechanism and a ground-state complex quenching mechanism.

13. The method of claim 7, wherein said detecting comprises acquiring a sequence of images of the fluorescently labeled γ

-phosphates.

14. The method of claim 1, wherein the fluorescently labeled γ

-phosphate is selectively detectable upon incorporation of the fluorescently labeled NTP into a polymerase extension product.

15. A method for genotyping or sequencing a single target nucleic acid molecule, said method comprising:

i) conducting a nucleic acid polymerase reaction in the presence of a single target nucleic acid, a primer, a nucleic acid polymerase and a plurality of fluorescently labeled nucleotide triphosphates (NTPs), the primer hybridizing to said target nucleic acid, wherein each type of nucleotide triphosphate (NTP) is differently fluorescently labeled on the γ

-phosphate , and wherein said nucleic acid polymerase reaction results in the production of a fluorescently labeled phosphate; and

ii) detecting each type of said fluorescently labeled phosphate individually to genotype or sequence said single target nucleic acid molecule.

16. The method of claim 15, wherein detecting each type of said fluorescently labeled phosphate comprises acquiring a sequence of images of said labeled phosphate.

17. The method of claim 15, wherein said method is for genotyping or sequencing an array of target nucleic acid sequences.

18. The method of claim 16, wherein said sequence of images is analyzed to track single molecules.

19. The method of claim 16, wherein said detecting is carried out by an evanescent optical field.