Apparatus and method for performing nucleic acid analysis

• US 9,709,503 B2
• Filed: 08/19/2016
• Issued: 07/18/2017
• 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: Korlach, Jonas, Turner, Stephen
• Primary Examiner(s): Chunduru, Prabha
• Application Number:
  US15/242,332
• Publication Number:
  US 20170002409A1
• Time in Patent Office: 333 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• CPC Class Codes: G01N 2021/6463 Optics
• CPC Class Codes: G01N 2021/7786 Fluorescence
• CPC Class Codes: G01N 21/00 Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light G01N3/00 - G01N19/00 take precedence
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/645 Specially adapted constructive features of fluorimeters
• CPC Class Codes: G01N 21/6452 Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 21/648 using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
• CPC Class Codes: G01N 21/77 by observing the effect on a chemical indicator
• CPC Class Codes: G01N 2201/06113 Coherent sources; lasers

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 method of sequencing a nucleic acid segment, comprising:

a) performing a single molecule nucleic acid sequencing reaction on a template nucleic acid molecule comprising a nucleic acid segment of interest, wherein the template nucleic acid molecule provides for iterative sequencing of the nucleic acid segment of interest, thereby generating multiple sequence reads of the nucleic acid segment of interest; and

b) analyzing the multiple sequence reads to determine a nucleic acid sequence for the nucleic acid segment of interest.

2. The method of claim 1, wherein the template nucleic acid molecule is circular and comprises one copy of the nucleic acid segment of interest.

3. The method of claim 1, wherein the template nucleic acid molecule comprises multiple copies of the nucleic acid segment of interest.

4. The method of claim 3, wherein the template nucleic acid molecule is circular.

5. The method of claim 3, wherein the template nucleic acid molecule is linear.

6. The method of claim 1, further comprising generating the template nucleic acid molecule, wherein the generating comprises:

obtaining a circular template comprising the nucleic acid segment of interest; and

performing a rolling circle amplification reaction on the circular template to generate the template nucleic acid molecule.

7. The method of claim 1, further comprising generating the template nucleic acid molecule, wherein the generating comprises:

generating multiple amplicons from a single template comprising the nucleic acid segment of interest; and

concatemerizing the multiple amplicons to generate the template nucleic acid molecule.

8. The method of claim 1, wherein the sequencing is performed using a template-directed polymerization reaction.

9. The method of claim 8, wherein a polymerase enzyme performing the template-directed polymerization reaction exhibits strand-displacement activity.

10. The method of claim 8, wherein a polymerase enzyme performing the template-directed polymerization reaction is a modified or unmodified Φ

29 DNA polymerase.

11. The method of claim 8, wherein the method is performed in the presence of a plurality of types of nucleotides or nucleotide analogs.

12. The method of claim 11, wherein each of the plurality of types of nucleotides or nucleotide analogs comprises a label.

13. The method of claim 12, wherein the label is attached at a base, sugar moiety, alpha phosphate, beta phosphate, or terminal phosphate of the nucleotides or nucleotide analogs.

14. The method of claim 12, wherein the label is a fluorescence resonance energy transfer donor or acceptor.

15. The method of claim 12, wherein the nucleotides or nucleotide analogs comprise a phosphate moiety selected from the group consisting of monophosphate, diphosphate, triphosphate, tetraphosphate, pentaphosphate, and hexaphosphate, and further comprise a label on a terminal phosphate of the phosphate moiety.

16. The method of claim 12, wherein each type of the plurality of types of nucleotides or nucleotide analogs has a different label that renders it distinguishable from every other type of the plurality of types during the generating of the multiple sequence reads, and wherein the generating comprises identifying each type of the plurality of types of nucleotides or nucleotide analogs based upon the different label.

17. The method of claim 1, wherein the single nucleic acid molecule is covalently or noncovalently attached to a support.

18. The method of claim 17, wherein the template nucleic acid molecule is attached to the support via interaction with at least one of the group consisting of:

a streptavidin/biotin linkage, an avidin/biotin linkage, a carbamate linkage, an ester linkage, an amide, a thiolester, an (N)-functionalized thiourea, a functionalized maleimide, an amino, a disulfide, an amide, a hydrazone linkage, an antibody, a silyl moiety, and an enzyme bound to the substrate.

19. The method of claim 1, wherein the sequencing takes place in an optical confinement.

20. The method of claim 19, wherein the optical confinement is a zero-mode waveguide.

21. The method of claim 19, wherein the optical confinement contains a single complex comprising the template nucleic acid molecule.

22. The method of claim 21, wherein the single complex further comprises a polymerase, and wherein the sequencing is performed using a template-directed polymerization reaction.

23. The method of claim 22, wherein the polymerase is a modified or unmodified Φ

29 DNA polymerase.

24. The method of claim 19, wherein the optical confinement is present in an array of optical confinements.