High throughput nucleic acid sequencing by expansion

• US 8,324,360 B2
• Filed: 03/30/2011
• Issued: 12/04/2012
• 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, Suryaprabha
• Application Number:
  US13/075,861
• Publication Number:
  US 20110237787A1
• Time in Patent Office: 615 Days
• Field of Search: None
• US Class Current: 536/23.1
• 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. An oligomer substrate construct for use in a template directed synthesis for sequencing a target nucleic acid, comprising a first probe moiety joined to a second probe moiety, each of the first and second probe moieties having an end group suitable for the template directed synthesis, and a tether having a first end and a second end with at least the first end of the tether joined to at least one of the first and second probe moieties, wherein the oligomer substrate construct when used in the template directed synthesis is capable of forming a daughter strand comprising a constrained Xpandomer, the constrained Xpandomer having a plurality of subunits coupled in a sequence corresponding to the contiguous nucleotide sequence of all or a portion of the target nucleic acid, wherein the individual subunits comprise a tether, the first and second probe moieties and at least one selectively cleavable bond that, upon cleavage, yields an Xpandomer of a length longer than the plurality of subunits of the daughter strand.

2. The oligomer substrate construct of claim 1 having the following structure:

3. The oligomer substrate construct of claim 1 having the following structure:

4. The oligomer substrate construct of claim 1 having the following structure:

5. The oligomer substrate construct of claim 1 having the following structure:

6. The oligomer substrate construct of claim 1 having the following structure:

7. An monomer substrate construct for use in a template directed synthesis for sequencing a target nucleic acid, comprising a nucleobase residue with end groups suitable for the template directed synthesis, and a tether having a first end and a second end with at least the first end of the tether joined to the nucleobase residue, wherein the monomer substrate construct when used in the template directed synthesis is capable of forming a daughter strand comprising a constrained Xpandomer, the constrained Xpandomer having a plurality of subunits coupled in a sequence corresponding to the contiguous nucleotide sequence of all or a portion of the target nucleic acid, wherein the individual subunits comprise a tether, the nucleobase residue and at least one selectively cleavable bond that, upon cleavage, yields an Xpandomer of a length longer than the plurality of subunits of the daughter strand.

8. The monomer substrate construct of claim 7 having the following structure:

9. The monomer substrate construct of claim 7 having the following structure:

10. The monomer substrate construct of claim 7 having the following structure:

12. The monomer substrate construct of claim 7 having the following structure:

31. The monomer substrate construct of claim 10 or the duplex daughter strand of claim 21, wherein the Xpandomer of a length longer than the plurality of subunits of the daughter strand has the following structure:

33. The monomer substrate construct of claim 12 or the duplex daughter strand of claim 23, wherein the Xpandomer of a length longer than the plurality of subunits of the daughter strand has the following structure:

11. A monomer substrate construct having the following structure:

32. The monomer substrate construct of claim 11 or the duplex daughter strand of claim 22, wherein the Xpandomer of a length longer than the plurality of subunits of the daughter strand has the following structure:

13. A duplex daughter strand for use in a template directed synthesis for sequencing a target nucleic acid, comprising a daughter strand duplexed with a template strand, the daughter strand comprising a constrained Xpandomer and having 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 that, upon cleavage, yields an Xpandomer of a length longer than the plurality of subunits of the daughter strand.

14. The duplex daughter strand of claim 13 having the following structure:

15. The duplex daughter strand of claim 13 having the following structure:

16. The duplex daughter strand of claim 13 having the following structure:

17. The duplex daughter strand of claim 13 having the following structure:

18. The duplex daughter strand of claim 13 having the following structure:

19. The duplex daughter strand of claim 13 having the following structure:

20. The duplex daughter strand of claim 13 having the following structure:

21. The duplex daughter strand of claim 13 having the following structure:

22. The duplex daughter strand of claim 13 having the following structure:

23. The duplex daughter strand of claim 13 having the following structure:

24. The oligomer substrate construct of claim 2 or the duplex daughter strand of claim 14, wherein the Xpandomer of a length longer than the plurality of subunits of the daughter strand has the following structure:

25. The oligomer substrate construct of claim 3 or the duplex daughter strand of claim 15, wherein the Xpandomer of a length longer than the plurality of subunits of the daughter strand has the following structure:

26. The oligomer substrate construct of claim 4 or the duplex daughter strand of claim 16, wherein the Xpandomer of a length longer than the plurality of subunits of the daughter strand has the following structure:

27. The oligomer substrate construct of claim 5 or the duplex daughter strand of claim 17, wherein the Xpandomer of a length longer than the plurality of subunits of the daughter strand has the following structure:

28. The oligomer substrate construct of claim 6 or the duplex daughter strand of claim 18, wherein the Xpandomer of a length longer than the plurality of subunits of the daughter strand has the following structure:

29. The monomer substrate construct of claim 8 or the duplex daughter strand of claim 19, wherein the Xpandomer of a length longer than the plurality of subunits of the daughter strand has the following structure:

30. The monomer substrate construct of claim 9 or the duplex daughter strand of claim 20, wherein the Xpandomer of a length longer than the plurality of subunits of the daughter strand has the following structure: