Apparatus and method for performing nucleic acid analysis

• US 7,476,503 B2
• Filed: 09/16/2005
• Issued: 01/13/2009
• Est. Priority Date: 09/17/2004
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Turner, Stephen, Korlach, Jonas
• Primary Examiner(s): Martinell; James
• Application Number:
  US11/228,376
• Publication Number:
  US 20060063264A1
• Time in Patent Office: 1,215 Days
• Field of Search: None
• US Class Current: 435/6.11
• CPC Class Codes: B01L 2200/0668 Trapping microscopic beads
• CPC Class Codes: B01L 2300/0654 Lenses; Optical fibres
• CPC Class Codes: B01L 2300/0896 Nanoscaled
• CPC Class Codes: B01L 3/5085 for multiple samples, e.g. microtitration plates
• CPC Class Codes: B82Y 20/00 Nanooptics, e.g. quantum optics or photonic crystals
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2521/543 Immobilised enzyme(s)
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer
• CPC Class Codes: G01N 21/6452 Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G02B 6/13 Integrated optical circuits characterised by the manufacturing method
• CPC Class Codes: Y10T 436/10 Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing

Abstract

The present invention relates to optical confinements, methods of preparing and methods of using them for analyzing molecules and/or monitoring chemical reactions. The apparatus and methods embodied in the present invention are particularly useful for high-throughput and low-cost single-molecular analysis.

Claims

1. A nucleic acid sequencing method comprising:

subjecting a target nucleic acid molecule to a template-directed polymerization reaction to yield a nascent nucleic acid strand that is complementary to the target nucleic acid molecule in the presence of a plurality of types of nucleotides or nucleotide analogs, and a polymerization enzyme exhibiting strand-displacement activity, wherein said template-directed polymerization reaction processes an identical sequence of nucleotides in the target nucleic acid molecule more than once;

registering a time sequence of incorporation of nucleotides or nucleotide analogs into the nascent nucleotide strand; and

determining a sequence of nucleotides in the target sequence.

2. The method of claim 1, wherein the target nucleic acid molecule is a circular nucleic acid.

3. The method of claim 2, wherein the target nucleic acid molecule is a circular DNA.

4. The method of claim 2, wherein said template-directed polymerization reaction processes a sequence of nucleotides in the circular nucleic acid multiple times.

5. The method of claim 1, wherein the target nucleic acid molecule comprises a sequence of nucleotides that is present multiple times within the target nucleic acid molecule.

6. The method of claim 1, wherein the target nucleic acid comprises a product of a template dependent replication of a circular nucleic acid.

7. Previously Presented) The method of claim 1, wherein the polymerization enzyme is a modified or unmodified φ

29 DNA polymerase.

8. The method of claim 1, wherein said target nucleic acid molecule is sequenced more than once by the polymerization enzyme.

9. The method of claim 1, wherein aid target nucleic acid molecule is sequenced more than twice by the polymerization enzyme.

10. The method of claim 1, wherein the nucleotides or nucleotide analogs further comprise a label.

11. The method of claim 10, wherein the label is attached to the nucleotides or nucleotide analogs at a base, sugar moiety, alpha phosphate, or beta phosphate, of said nucleotides or nucleotide analogs.

12. The method of claim 10, wherein the label is attached to the nucleotides or nucleotide analogs at a terminal phosphate.

13. The method of claim 10, wherein the label is on a terminal phosphate of a nucleotide or nucleotide analog, comprising a phosphate moiety selected from the group consisting of monophosphate, diphosphate, triphosphate, tetraphosphate, pentaphosphate, and hexaphosphate.

14. The method of claim 10, wherein each type of the nucleotides or nucleotide analogs has a different label which is distinguished from one another during said registering step.

15. The method of claim 10, wherein the label is a fluorescence resonance energy transfer donor or acceptor.

16. The method of claim 1, wherein said target nucleic acid molecule is attached to a support.

17. The method of claim 1, wherein said target nucleic acid molecule is hybridized to primer which is attached to a support.

18. The method of claim 1, wherein said polymerization enzyme is attached to a support.

19. The method of claim 1, wherein said registering is performed while the template-directed polymerization reaction is taking place.

20. The method of claim 1, wherein said registering step comprising identifying nucleotides or nucleotide analogs provided in said plurality of types of nucleotides or nucleotide analogs.

21. The method of claim 1, wherein the template-directed polymerization reaction takes place in an optical confinement.

22. The method of claim 1, wherein the template-directed polymerization reaction takes place in an array of optical confinements.

23. The method of claim 1, wherein the template-directed polymerization reaction takes place in a zero-mode waveguide.

24. The method of claim 1, wherein the target nucleic acid molecule and the polymerization enzyme form a single complex immobilized in an optical confinement.

25. The method of claim 24, wherein the array of optical confinements has a fill fraction greater than 0.0001.

26. The method of claim 24, wherein the array of optical confinements has a fill fraction greater than 0.01.

27. The method of claim 24, wherein the array of optical confinements has a fill fraction between 0.00 and 0.1

28. The method of claim 24, wherein the array of optical confinements has a fill fraction greater than 0.1.

29. The method of claim 22, wherein the array of optical confinements has a density on a substrate exceeding 4 ×

10⁴ confinements per mm².

30. The method of claim 22, wherein the array of optical confinements has a density on a substrate exceeding 10⁵ confinements per mm².