High throughput nucleic acid sequencing by expansion

• US 8,349,565 B2
• Filed: 03/30/2011
• Issued: 01/08/2013
• Est. Priority Date: 06/19/2007
• Status: Active Grant

• Current Assignee: Roche Sequencing Solutions, Inc. (Roche Holding AG)
• Original Assignee: Stratos Genomics Inc.
• Inventors: Kokoris, Mark Stamatios, McRuer, Robert N.
• Primary Examiner(s): Chunduru, Prabha
• Application Number:
  US13/075,864
• Publication Number:
  US 20110251079A1
• Time in Patent Office: 650 Days
• Field of Search: None
• US Class Current: 435/6.12
• 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 1/68 involving nucleic acids
• 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 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters
• CPC Class Codes: C12Q 2523/107 Chemical cleaving agents
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore

Abstract

Nucleic acid sequencing methods and related products are disclosed. Methods for sequencing a target nucleic acid comprise providing a daughter strand produced by a template-directed synthesis, the daughter strand comprising a plurality of subunits coupled in a sequence corresponding to a contiguous nucleotide sequence of all or a portion of the target nucleic acid, wherein the individual subunits comprise a tether, at least one probe or nucleobase residue, and at least one selectively cleavable bond. The selectively cleavable bond(s) is/are cleaved to yield an Xpandomer of a length longer than the plurality of the subunits of the daughter strand, the Xpandomer comprising the tethers and reporter elements for parsing genetic information in a sequence corresponding to the contiguous nucleotide sequence of all or a portion of the target nucleic acid. Reporter elements of the Xpandomer are then detected. Corresponding products, including Xpandomers and oligomeric and monomeric substrate constructs are also disclosed.

Claims

1. A method for sequencing a target nucleic acid, comprising:

a) providing a daughter strand produced by a template-directed synthesis, the daughter strand comprising a plurality of subunits coupled in a sequence corresponding to a contiguous nucleotide sequence of all or a portion of the target nucleic acid, wherein the daughter strand is formed from a plurality of oligomer or monomer substrate constructs, wherein the individual subunits of the daughter strand comprise a tether, at least one probe or nucleobase residue, and at least one selectively cleavable bond, wherein the tether is incorporated into the individual subunits of the daughter strand after formation of the daughter strand from the plurality of oligomer or monomer substrate constructs, and wherein the tether, upon cleavage of the at least one cleavable bond, permits lengthening of the subunits of the daughter strand;

b) cleaving the at least one selectively cleavable bond to yield an Xpandomer of a length longer than the plurality of the subunits of the daughter strand, the Xpandomer comprising the tethers and reporter elements for parsing genetic information in a sequence corresponding to the contiguous nucleotide sequence of all or a portion of the target nucleic acid; and

c) detecting the reporter elements of the Xpandomer.

2. The method of claim 1, wherein the reporter elements for parsing the genetic information are associated with the tethers of the Xpandomer.

3. The method of claim 1, wherein the reporter elements for parsing genetic information are associated with the daughter strand prior to cleavage of the at least one selectively cleavable bond.

4. The method of claim 1, wherein the reporter elements for parsing genetic information are associated with the Xpandomer after cleavage of the at least one selectively cleavable bond.

5. The method of claim 1, wherein the Xpandomer further comprises all or a portion of the at least one probe or nucleobase residue.

6. The method of claim 5, wherein the reporter elements for parsing the genetic information are or are associated with the at least one probe or nucleobase residue.

7. The method of claim 1, wherein the at least one selectively cleavable bond is a covalent bond.

8. The method of claim 1, wherein the at least one selectively cleavable bond is an intra-tether bond.

9. The method of claim 1, wherein the at least one selectively cleavable bond is a bond between or within probes or nucleobase residues of the daughter strand.

10. The method of claim 1, wherein the at least one selectively cleavable bond is a bond between the probes or nucleobase residues of the daughter strand and a target template.

11. The method of claim 1, wherein the Xpandomer comprises the following structure:

12. The method of claim 11, wherein the daughter strand, prior to cleavage of the at least one selectively cleavable bond, comprises a template-daughter strand duplex having the following structure:

13. The method of claim 1, wherein the Xpandomer comprises the following structure:

14. The method of claim 13, wherein the daughter strand, prior to cleavage of the at least one selectively cleavable bond, comprises a template-daughter strand duplex having the following structure:

15. The method of claim 1, wherein the Xpandomer comprises the following structure:

16. The method of claim 15, wherein the daughter strand, prior to cleavage of the at least one selectively cleavable bond, comprises a template-daughter strand duplex having the following structure:

17. The method of claim 1, wherein the Xpandomer comprises the following structure:

18. The method of claim 17, wherein the daughter strand, prior to cleavage of the at least one selectively cleavable bond, comprises a template-daughter strand duplex having the following structure:

19. The method of claim 1, wherein the Xpandomer comprises the following structure:

20. The method of claim 19, wherein the daughter strand, prior to cleavage of the at least one selectively cleavable bond, comprises a template-daughter strand duplex having the following structure:

21. The method of claim 1, wherein the Xpandomer comprises the following structure:

22. The method of claim 21, wherein the daughter strand, prior to cleavage of the at least one selectively cleavable bond, comprises a template-daughter strand duplex having the following structure:

23. The method of claim 1, wherein the Xpandomer comprises the following structure:

24. The method of claim 23, wherein the daughter strand, prior to cleavage of the at least one selectively cleavable bond, comprises a template-daughter strand duplex having the following structure:

25. The method of claim 1, wherein the Xpandomer comprises the following structure:

26. The method of claim 25, wherein the daughter strand, prior to cleavage of the at least one selectively cleavable bond, comprises a template-daughter strand duplex having the following structure:

27. The method of claim 1, wherein the Xpandomer comprises the following structure:

28. The method of claim 27, wherein the daughter strand, prior to cleavage of the at least one selectively cleavable bond, is duplexed with a template strand to yield a duplex daughter strand having the following structure:

29. The method of claim 1, wherein the Xpandomer comprises the following structure:

30. The method of claim 29, wherein the daughter strand, prior to cleavage of the at least one selectively cleavable bond, is duplexed with a template strand to yield a duplex daughter strand having the following structure: