Efficient base determination in sequencing reactions

Patent metadata

Field	Value
Publication number	US-9267172-B2
Application number	US-201313965166-A
Country	US
Kind code	B2
Filing date	Aug 12, 2013
Priority date	Nov 5, 2007
Publication date	Feb 23, 2016
Grant date	Feb 23, 2016

Abstract

The present invention is directed to compositions and methods for nucleic acid identification and detection. Compositions and methods of the present invention include extracting and fragmenting target nucleic acids from a sample, using the fragmented target nucleic acids to produce target nucleic acid templates and subjecting those target nucleic acid templates to amplification methods to form nucleic acid nanoballs. The invention also includes methods of detecting and identifying sequences using various sequencing applications, including sequencing by ligation methods.

First claim

What is claimed:

1. A method for identifying a first nucleotide at a detection position of a target sequence comprising a plurality of detection positions, said method comprising:
(a) providing a plurality of nucleic acid constructs, wherein each nucleic acid construct comprises a plurality of monomers and each monomer comprises:
i) a first target domain of said target sequence comprising a first set of target detection positions; and
ii) a first adaptor comprising an anchor site;

b) hybridizing a first anchor probe to said anchor site;
c) hybridizing a second anchor probe to a sequence to adjacent to and contiguous with the anchor site, wherein said second anchor probe is partly or entirely degenerate and the hybridized second anchor probe can be ligated to the hybridized first anchor probe;
d) hybridizing one, two, or three additional anchor probes to the target sequence between the first target domain and the site of hybridization of the second anchor probe;

wherein said additional anchor probe(s) are fully degenerate;
e) hybridizing at least a first sequencing probe to said first target domain,

wherein said first sequencing probe comprises:
i) a first probe domain complementary to said target domain;
ii) a unique nucleotide at a first interrogation position; and
iii) a label;
under conditions wherein said sequencing probe hybridizes to said target domain if said unique nucleotide is complementary to a first nucleotide in said first set of target detection positions;
wherein said hybridized first anchor probe, second anchor probe, additional anchor probe(s) and first sequencing probe are hybridized to adjacent sequences and can be ligated together to form a ligation product; and
f) ligating said anchor probes and said sequencing probe to form a probe ligation product; and then
g) detecting said probe ligation product thereby identifying said first nucleotide.

2. The method according to claim 1 wherein the nucleic acid constructs are disposed on a surface.

3. The method according to claim 2 wherein each nucleic acid construct is a concatemer that comprises a plurality of monomers, and each monomer comprises the target domain and the adaptor.

4. The method according to claim 3 wherein said surface is functionalized.

5. The method according to claim 4 wherein said functionalized surface comprises functional moieties selected from the group consisting of amines, silanes, and hydroxyls.

6. The method according to claim 3 wherein said surface comprises a plurality of spatially distinct regions comprising said immobilized concatemers.

7. The method of claim 6 wherein the spatially distinct regions are arranged as a regular array.

8. The method according to claim 6 wherein said concatemers are non-covalently attached to the surface.

9. The method according to claim 3 wherein said concatemers are immobilized on said surface using capture probes.

10. The method according to claim 3 wherein each of said monomer comprises a plurality of adaptors.

11. The method according to claim 1 wherein steps b)-g) are carried out for multiple cycles, thereby identifying nucleotides at additional target detection positions in said set of target detection positions, wherein in different cycles different sequencing probes are used, wherein said different sequencing probes differ by having a unique nucleotide at different interrogation positions.

12. The method according to claim 1 , wherein the additional anchor probe that is ligated to the first anchor probe comprises a set of second anchor probes comprising at least three degenerate bases that hybridize to said sequences outside said second anchor site.

13. The method according to claim 1 , wherein said second anchor probe comprises at least one terminus that is selectively activatable for ligation.

14. The method according to claim 1 further comprising fragmenting genomic nucleic acid to form target sequences.

15. The method according to claim 1 wherein said target sequence is a genomic nucleic acid sequence.

16. The method according to claim 15 wherein said genomic nucleic acid sequences are human.

17. The method of claim 1 wherein step (d) comprises hybridizing one additional anchor probe to the target sequence.

18. The method of claim 1 wherein step (d) comprises hybridizing two additional anchor probes to the target sequence.

19. The method of claim 1 wherein step (d) comprises hybridizing three additional anchor probes to the target sequence.

Assignees

• Complete Genomics Inc

Inventors

• Drmanac Radoje
• Callow Matthew J

Classifications

• C12Q1/6874 Primary

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• C12Q1/6855

  Ligating adaptors · CPC title