Nucleic acid sample enrichment for sequencing applications

• US 8,728,764 B2
• Filed: 10/02/2009
• Issued: 05/20/2014
• Est. Priority Date: 10/02/2008
• Status: Active Grant

• Current Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Original Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Inventors: Boutell, Jonathan Mark
• Primary Examiner(s): Wilder, Cynthia B
• Application Number:
  US13/119,131
• Publication Number:
  US 20110172119A1
• Time in Patent Office: 1,691 Days
• Field of Search: 435/91.2
• US Class Current: 435/91.2
• 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 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides

Abstract

The present invention relates to the field of molecular biology, and more specifically to methods for reducing the complexity of a nucleic acid sample.

Claims

1. A method for selectively hybridizing a portion of a nucleic acid sample comprising:

amplifying a pool of oligonucleotide probes on a solid support to generate probe amplicons and

after said amplifying, applying a nucleic acid sample comprising genomic DNA to said solid support to selectively hybridize a portion of the nucleic acid sample to the probe amplicons.

2. The method according to claim 1 wherein each member of the pool of oligonucleotide probes comprises:

(i) a universal sequence at the 3′

end that is common to the probes in the pool,

(ii) a first known region capable of hybridizing to a first pre-selected region of a nucleic acid sequence of interest,

(iii) a second known region capable of hybridizing to a second pre-selected region of the nucleic acid sequence of interest, and

(iv) a universal sequence at the 5′

end that is common to the probes in the pool.

3. The method according to claim 2 wherein each member of the pool of oligonucleotide probes further comprises a cleavage site between the first and second known regions.

4. The method according to claim 1 wherein the method comprises:

a) providing a probe library comprising a pool of oligonucleotides of different sequences, wherein each member of the probe library comprises a universal sequence at the 3′

end that is common to the probes in the library, a first known region capable of hybridizing to a pre-selected region of the nucleic acid sample of interest, a second known region capable of hybridizing to a second pre-selected region of the nucleic acid sample in the vicinity of the first known sequence, a universal sequence at the 5′

end that is common to the probes in the library, and a site for selectively cleaving the oligonucleotide strand between the first and second known regions;

b) separating the individual molecules of the probe library and amplifying the separated individual molecules in parallel to form a collection of probe amplicons where each member of the collection contains multiple copies of a single sequence;

c) cleaving the probe amplicons to disconnect the first and second known regions; and

d) hybridizing the nucleic acid sample to the first known regions.

5. The method according to claim 4 wherein the method further comprises:

e) extending the first known regions using the hybridized nucleic acids as templates to produce extended immobilized nucleic acid strands wherein an extended strand comprises a region complementary to the second known region;

f) removing the nucleic acid sample from the extended strands;

g) hybridizing the extended strands to the oligonucleotides comprising the second known region;

h) extending the second known region to form a duplex immobilized at both ends comprising both the first and second known regions; and

i) amplifying the duplexes.

6. The method according to claim 5 wherein the resultant amplified duplexes are sequenced to obtain sequence information on a region between the first and second known sequences.

7. The method according to claim 4 wherein the method further comprises:

e) extending the first known regions using the nucleic acid samples as templates;

f) joining the 3′

ends of the extended strands to the 5′

ends of the second known region to generate immobilized extended strands comprising the first and second regions; and

g) amplifying the immobilized extended strands.

8. The method according to claim 7 wherein the resultant amplified strands are sequenced to obtain sequence information on a region between the first and second known sequences.

9. The method of claim 1, wherein the probe library comprises greater than 10³ different sequences.

10. The method of claim 9, wherein the probe library comprises greater than 10⁶ different sequences.

11. The method of claim 3 wherein the cleavage site is a recognition site for a restriction endonuclease.

12. The method of claim 11 wherein the restriction endonuclease is a remote cutting restriction endonuclease.

13. The method of claim 1 wherein the solid support is a planar surface or a population of beads.

14. A method of selecting and amplifying polynucleotides on a solid support, comprising:

a) providing a plurality of amplification oligonucleotides immobilized on a solid support;

b) hybridizing a population of oligonucleotide probes to a subset of said amplification oligonucleotides, each of said oligonucleotide probes comprising a first portion which is complementary to the amplification oligonucleotides and a second portion which comprises sequence from a selected region of a template polynucleotide;

c) performing an extension reaction to extend hybridized amplification oligonucleotides to produce a population of support-bound capture oligonucleotides, each capture oligonucleotide in said population comprising a sequence that is complementary to a selected region of a template polynucleotide;

d) applying a population of template polynucleotides to the solid support under conditions such that the template polynucleotides selectively hybridise to the support-bound capture oligonucleotides;

e) extending the support-bound capture oligonucleotides that are hybridized to the template polynucleotides, thereby generating extension products complementary to the template polynucleotides; and

f) amplifying the extension products, wherein the amplifying comprises annealing one or more of the immobilised amplification oligonucleotides to one or more of the extension products, thereby producing a solid-phase amplification product.

15. The method of claim 14, further comprising:

g) sequencing a first strand of the solid-phase amplification product of step f) to obtain at least part of the nucleotide sequence of the template polynucleotide.

16. The method of claim 14 wherein the amplification is isothermal.

17. The method of claim 14 wherein one end of the template polynucleotides comprises an adapter sequence.

18. The method of claim 17, wherein the template polynucleotides in the nucleic acid sample comprise different sequences and the adapter sequence is the same for each template polynucleotides.

19. The method of claim 14, wherein the template polynucleotides comprise PCR amplicons.

20. The method of claim 14, wherein the template polynucleotides comprise genomic DNA fragments.