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

• US 6,762,048 B2
• Filed: 05/14/2001
• Issued: 07/13/2004
• 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): Whisenant, Ethan
• Assistant Examiner(s): Lu, Frank Wei Min
• Application Number:
  US09/859,104
• Publication Number:
  US 20020115076A1
• Time in Patent Office: 1,156 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/183, 435/283.1, 435/287.1, 435/287.2, 435/288.4, 435/288.5, 436/501, 436/94, 536/23.1, 536/24.3, 536/24.33, 536/25.32, 422/50, 422/68.1
• US Class Current: 435/287.1
• 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. An apparatus suitable for sequencing a target nucleic acid molecule comprising:

a microstructure defining a flow chamber containing

(a) a support, and

(b) only one single individual immobilized molecule in single molecule configuration, said immobilized molecule selected from the group consisting of a nucleic acid polymerizing enzyme and said target nucleic acid;

which microstructure is configured to permit labeled nucleotide analogs that are not positioned on the support to move through the flow chamber.

2. An apparatus of claim 1, wherein said immobilized molecule is a nucleic acid polymerizing enzyme.

3. An apparatus of claim 1, wherein said immobilized molecule is a target nucleic acid.

4. An apparatus of claim 1, wherein said flow chamber is of plastic or glass.

5. An apparatus of claim 1, further comprising a detector capable of imaging a single molecule.

12. An apparatus of claim 1, 6 or 11, wherein said single molecule configuration represents an array of molecules where members of the array are present as an individual molecule each located in a defined location.

13. An apparatus of claim 12, wherein each member of said array is different.

14. An apparatus of claim 12, wherein each member is so spatially resolved that it is detected as an individual molecule.

15. An apparatus of claim 12, wherein said array of molecules is addressable.

16. An apparatus of claim 1, 6 or 11, said apparatus further comprising a detector 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 coupled 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.

17. An apparatus of claim 16, wherein said detecting comprises acquiring a sequence of images.

18. An apparatus of claim 16, wherein the sequence of images is analyzed to track single molecules.

19. An apparatus of claim 16, wherein said detecting is carried out by a mechanism that provides low background and enables the detection of single molecules.

20. An apparatus of claim 16, wherein said detecting is carried out by an evanescent optical field.

21. An apparatus of claim 16, wherein said detecting is carried out by a mechanism selected from the group consisting of fluorescence resonance energy transfer, an electron transfer mechanism, an excited-state lifetime mechanism and a ground-state complex quenching mechanism.

6. An apparatus suitable for sequencing a target nucleic acid molecule comprising:

a microstructure defining a flow chamber, said flow chamber divided into separate channels, each of which contains

(a) a support, and

(b) only one single individual immobilized molecule in single molecule configuration, said immobilized molecule selected from the group consisting of a nucleic acid polymerizing enzyme and said target nucleic acid;

which microstructure is configured to permit labeled nucleotide analogs that are not positioned on the support to move through the flow chamber.

7. An apparatus of claim 6, wherein said immobilized molecule is a nucleic acid polymerizing enzyme.

8. An apparatus of claim 6, wherein said immobilized molecule is a target nucleic acid.

9. An apparatus of claim 6, wherein said flow chamber is of plastic or glass.

10. An apparatus of claim 6, further comprising a detector capable of imaging a single molecule.

11. An apparatus suitable for sequencing a target nucleic acid molecule comprising:

a microstructure defining a flow chamber containing

(a) a support having a fixed charge,

(b) only one single individual immobilized molecule in single molecule configuration, said immobilized molecule selected from the group consisting of a nucleic acid polymerizing enzyme and said target nucleic acid, and

(c) two electrodes positioned so as to provide a voltage gradient across said microstructure;

which microstructure is configured to permit labeled nucleotide analogs that are not positioned on the support to move through the flow chamber.