Bi-directional sequencing compositions and methods

• US 9,139,874 B2
• Filed: 07/06/2012
• Issued: 09/22/2015
• Est. Priority Date: 07/07/2011
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Chen, Zhoutao, Nikiforov, Theo, Myers, Jason, Dressman, Devin
• Primary Examiner(s): HORLICK, KENNETH R
• Application Number:
  US13/543,521
• Publication Number:
  US 20130012399A1
• Time in Patent Office: 1,173 Days
• Field of Search: 435/91.1
• US Class Current: 1/1
• 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: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/307 Single strand endonuclease
• CPC Class Codes: C12Q 2521/325 Single stranded exonuclease
• CPC Class Codes: C12Q 2525/125 incorporating agents resulting in resistance to degradation
• CPC Class Codes: C12Q 2535/119 Double strand sequencing
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target

Abstract

In some embodiments, methods for obtaining sequence information from a nucleic acid template linked to a support include hybridizing a first primer to a template strand linked to a support, sequencing a portion of the nucleic acid template, thereby forming an extended first primer product that is complementary to a portion of the nucleic acid template. In some embodiments, the method further includes introducing a nick into a portion of the template strand that is hybridized to the extended first primer product, degrading a portion of the template strand from the nick using a degrading agent, where a portion of the extended first primer remains hybridized to an undegraded portion of the template strand, and sequencing at least some of the single-stranded portion of the extended first primer by synthesis.

Claims

1. A method for obtaining sequence information from a portion of a nucleic acid template, comprising:

hybridizing a first primer to a template strand;

sequencing a portion of the nucleic acid template, wherein the sequencing includes extending the first primer, thereby forming an extended first primer product that is complementary to a portion of the nucleic acid template;

introducing a nick into a portion of the template strand that is hybridized to the extended first primer product;

wherein the nick includes a free 5′

end and a free 3′

end in the template strand;

degrading a portion of the template strand from the free 5′

end of the nick using a degrading agent, thereby generating a single-stranded portion of the extended first primer product, wherein a portion of the extended first primer product remains hybridized to an undegraded portion of the template strand; and

sequencing at least some of the single-stranded portion of the extended first primer product.

2. The method of claim 1, wherein the template strand is linked to a support through the 5′

end of the template strand.

3. The method of claim 1, wherein the template strand is linked to a support through at least one nucleotide in the template strand that is situated 5′

of the nick site in the template strand.

4. The method of claim 3, wherein at least one nucleotide in the template strand situated 5′

of the nick site is resistant to the degrading agent.

5. The method of claim 4, wherein the at least one nucleotide resistant to the degrading agent comprises a phosphorothioate, a 2-O-Methyl RNA residue, or a combination thereof.

6. The method of claim 1, wherein the first primer is resistant to degradation by the degrading agent.

7. The method of claim 1, wherein the extended first primer product is resistant to degradation by the degrading agent.

8. The method of claim 1, wherein the degrading agent includes a 5′

-3′

exonuclease, and the degrading further includes digesting the template strand from the free 5′

end of the nick using the 5′

-3′

exonuclease.

9. The method of claim 1, wherein sequencing at least some of the single-stranded portion of the extended first primer includes extending the free 3′

end of the nick via nucleic acid synthesis, thereby synthesizing a nucleic acid molecule that is complementary to at least some of the single-stranded portion of the extended first primer.

10. The method of claim 1, wherein sequencing at least some of the single-stranded portion of the extended first primer product includes hybridizing a reverse primer to a sequence within the single-stranded portion of the extended first primer product, and extending the reverse primer using a polymerase.

11. A method for nucleic acid sequencing comprising:

hybridizing a first primer to a distal end of a nucleic acid strand having a distal and proximal end, wherein the proximal end of the nucleic acid strand is linked to a support;

extending the hybridized first primer in the direction of the proximal end of the nucleic acid strand and the support, thereby forming an extended first primer product that is complementary to a portion of the nucleic acid strand and obtaining a first sequencing read;

introducing a site-specific nick into the proximal end of the nucleic strand hybridized to the extended first primer product;

degrading a portion of the nucleic acid strand, thereby generating a single-stranded portion within the extended first primer product, wherein a portion of the extended first primer product remains hybridized to the nucleic acid strand; and

extending the single-stranded portion within the extended first primer product, thereby obtaining a second sequencing read.

12. The method of claim 11, wherein the extending is performed via template-dependent nucleic acid synthesis.

13. The method of claim 11, wherein the first primer or the extended first primer product is nuclease resistant.

14. The method of claim 11, wherein the support is an Ion Sphere Particle.

15. The method of claim 11, wherein the nucleic acid sequencing is label-free or ion based sequencing.

16. The method of claim 11, wherein the site-specific nick is introduced by a restriction enzyme or a uracil DNA glycosylase.

17. The method of claim 11, further comprising consolidating the first and second sequencing read.

18. The method of claim 17, wherein consolidating comprises aligning the first and second sequencing read against a reference sequence.

19. The method of claim 18, wherein aligning the first and second sequencing reads against a reference sequence determines the presence of a deletion, insertion, translocation, inversion, variation, mutation or mismatch in the nucleic acid strand.

20. The method of claim 11, wherein the extended first primer product is cross-linked to the support.

21. The method of claim 20, wherein the extended first primer product is cross-linked to the support prior to introducing the site-specific nick or the degrading step.

22. The method of claim 20, wherein the extended first primer product is cross-linked to the support by a photo-chemical reaction.

23. A method for obtaining bi-directional sequence information from a nucleic acid molecule, comprising:

hybridizing a first primer to a nucleic acid molecule;

sequencing a portion of the nucleic acid molecule, wherein the sequencing includes extending the first primer, thereby forming an extended first primer product that is complementary to a portion of the nucleic acid molecule;

introducing a nick into a portion of the nucleic acid molecule that is hybridized to the extended first primer product;

wherein the nick includes a free 5′

end and a free 3′

end in the nucleic acid molecule;

degrading a portion of the nucleic acid molecule from the free 5′

end of the nick using a degrading agent, thereby generating a single-stranded portion of the extended first primer product, wherein a portion of the extended first primer product remains hybridized to an undegraded portion of the nucleic acid molecule; and

sequencing at least some of the single-stranded portion of the extended first primer product.

24. The method of claim 23, wherein the nucleic acid molecule is linked to a support.

25. The method of claim 23, wherein the nucleic acid molecule comprises a single stranded template.

26. The method of claim 23, wherein the sequencing comprises sequencing by synthesis, sequencing by ligation or sequencing by hybridization.

27. A method for improving nucleic acid sequencing accuracy comprising:

hybridizing a first primer to a distal end of a nucleic acid strand having a distal and proximal end, wherein the proximal end of the nucleic acid strand is linked to a solid support;

extending the hybridized first primer in the direction of the proximal end of the nucleic acid strand and the solid support, thereby forming an extended first primer product that is complementary to a portion of the nucleic acid strand and obtaining a first sequencing read;

introducing a site-specific nick into the proximal end of the nucleic strand hybridized to the extended first primer product;

degrading a portion of the nucleic acid strand, thereby generating a single-stranded portion within the extended first primer product, wherein a portion of the extended first primer product remains hybridized to the nucleic acid strand; and

extending the single-stranded portion within the extended first primer product, thereby obtaining a second sequencing read; and

aligning the first and second sequencing read to obtain a nucleic acid sequence with improved accuracy.