Preparations, compositions, and methods for nucleic acid sequencing

• US 8,795,961 B2
• Filed: 09/03/2009
• Issued: 08/05/2014
• Est. Priority Date: 09/05/2008
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Rank, David R., Peluso, Paul
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): THOMAS, DAVID C
• Application Number:
  US12/553,478
• Publication Number:
  US 20100081143A1
• Time in Patent Office: 1,797 Days
• Field of Search: None
• US Class Current: 435/6.1
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer

Abstract

Preparations, compositions and methods of sequencing nucleic acids are provided. The preparations, methods and compositions employ multiple priming sites on a target nucleic acid and typically utilize single molecule sequencing processes to identify sequence portions of the target which are then assembled to obtain the sequence of the target.

Claims

1. A method of sequencing nucleic acids, comprising;

a) providing a sample comprising target nucleic acids;

b) adding a set of primers to said sample, wherein the set comprises primers having different nucleotide compositions;

c) contacting the target nucleic acids in the sample with the set of primers under conditions that promote hybridization to generate a set of target-primer complexes;

d) using a polymerase enzyme, pre-extending primers in the target-primer complexes to generate pre-extended target-primer complexes that further comprise the polymerase enzyme;

e) confining each of the pre-extended target-primer complexes to a separate reaction site on a substrate comprising a plurality of reaction sites;

f) initiating template-directed synthesis of nascent nucleic acid strands from each of the pre-extended target-primer complexes, wherein the nascent nucleic acid strands are complementary to the target nucleic acids, and wherein the template-directed synthesis is performed by the polymerase enzyme;

g) detecting processive incorporation of nucleotides into each of the nascent nucleic acid strands with no intervening buffer exchange during the template-directed synthesis, wherein the detecting further identifies nucleotide sequence reads for the nascent nucleic acid strands; and

h) based upon the results of step f, determining a nucleotide sequence of the target nucleic acids.

2. The method of claim 1, wherein each of the plurality of reaction sites is positioned such that a signal emitted from any one reaction site is optically resolvable from any other signals emitted from any other of the plurality of reaction sites.

3. The method of claim 2, wherein the substrate comprises a plurality of optical confinements, each of which contains a single one of the plurality of reaction sites.

4. The method of claim 1, wherein the confining is performed through immobilization of each one of the target nucleic acids within the pre-extended target-primer complexes to the substrate.

5. The method of claim 4, wherein the immobilization comprises hybridization of each one of the target nucleic acids within the pre-extended target-primer complexes to a complementary oligonucleotide bound to the substrate.

6. The method of claim 4, wherein the immobilization comprises binding the polymerase enzyme within each of the pre-extended target-primer complexes to the substrate.

7. The method of claim 1, wherein the template-directed synthesis is catalyzed by an enzyme selected from the group consisting of a DNA polymerase, an RNA polymerase, and a reverse transcriptase.

8. The method of claim 7, wherein the enzyme is capable of strand displacement.

9. The method of claim 7, wherein the enzyme is thermostable.

10. The method of claim 7, wherein the enzyme has a processivity of at least 500 base pairs.

11. The method of claim 1, wherein the detecting comprises detecting signals from single target-primer complexes that further comprise the polymerase enzyme, wherein the signals are indicative of the identity of a nucleotide incorporated during the template-directed synthesis.

12. The method of claim 1, wherein the set of primers is a set of random primers.

13. The method of claim 1, wherein the set of primers is designed to anneal to selected regions of the target nucleic acids.

14. The method of claim 1, wherein the target nucleic acids are not subjected to amplification prior to said template-directed synthesis.

15. The method of claim 1, wherein the target nucleic acids are not subjected to cloning prior to said template-directed synthesis.

16. The method of claim 1, wherein the target nucleic acids are subjected to fragmentation prior to said template-directed synthesis.

17. The method of claim 1, wherein the nucleotide sequence of the target nucleic acids is not known prior to said template-directed synthesis.

18. The method of claim 1, wherein the target nucleic acids comprise genomic DNA.

19. The method of claim 1, wherein the target nucleic acids comprise RNA.

20. The method of claim 1, wherein the target nucleic acids are circular nucleic acids.

25. The method of claim 1, wherein the target nucleic acids are circular nucleic acid templates that are repeatedly processed in the sequence-by-synthesis reactions to generate multiple copies of the nucleotide sequences of the nucleic acid templates for use in step h.

27. The method of claim 1, wherein the pre-extension is performed in the presence of at least three different dNTPs or analogs thereof.

21. A method of generating sequence information for a target nucleic acid, comprising:

a) providing a plurality of nucleic acid templates corresponding to the target nucleic acid;

b) providing multiple primers that anneal to the nucleic acid templates;

c) annealing the multiple primers to multiple locations on the nucleic acid templates;

d) contacting the nucleic acid templates with single polymerase enzymes that bind at single primers annealed to the nucleic acid templates to generate primer-template-polymerase complexes;

e) using the polymerase enzymes, pre-extending primers in the primer-template-polymerase complexes to generate pre-extended primer-template-polymerase complexes;

f) confining each of the pre-extended primer-template-polymerase complexes to separate reaction sites on a substrate comprising a plurality of reaction sites;

g) performing sequence-by-synthesis reactions to determine nucleotide sequences of the nucleic acid templates within the pre-extended primer-template-polymerase complexes, wherein the performing comprises identifying bases complementary to the nucleic acid templates by detecting labels that are released from said bases upon incorporation of said bases into nascent strands complementary to the nucleic acid templates; and

h) assembling an overall nucleotide sequence of the target nucleic acid from the nucleotide sequences of the nucleic acid templates.

22. The method of claim 21, wherein the plurality of nucleic acid templates corresponding to the target nucleic acid comprises multiple overlapping fragments with nucleotide sequences identical to portions of the target nucleic acid.

23. The method of claim 21, wherein the nucleic acid templates are circular nucleic acid templates that are repeatedly processed in the sequence-by-synthesis reactions to generate multiple copies of the nucleotide sequences of the nucleic acid templates for use in step h.

24. The method of claim 21, wherein the nucleic acid templates are linear nucleic acids and the method further comprises repeating steps c, d, e, f, and g multiple times to determine multiple nucleotide sequences from each of the nucleic acid templates downstream of multiple single primers annealed to the nucleic acid templates for use in step h.

26. The method of claim 21, wherein the confining comprises binding the polymerase enzymes within the pre-extended primer-template-polymerase complexes to the separate reaction sites on the substrate.

28. The method of claim 21, wherein the pre-extension is performed in the presence of at least three different dNTPs or analogs thereof.