NUCLEIC ACID SAMPLE ENRICHMENT FOR SEQUENCING APPLICATIONS

• US 20110172119A1
• Filed: 10/02/2009
• Published: 07/14/2011
• 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
• Granted Patent: US 8,728,764 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• 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 selecting a portion of a nucleic acid sample comprising amplifying a pool of oligonucleotide probes on a solid support to generate probe amplicons and hybridising 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 hybridising to a first pre-selected region of a nucleic acid sequence of interest,

(iii) a second known region capable of hybridising 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 pool of oligonucleotides of different sequences, wherein each member of the probe pool comprises a universal sequence at the 3′

end that is common to the probes in the pool, a first known region capable of hybridising to a pre-selected region of the nucleic acid sample of interest, a second known region capable of hybridising 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 pool, and a site for selectively cleaving the oligonucleotide strand between the first and second known regions;

b) separating the individual molecules of the probe pool 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) hybridising 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 hybridised nucleic acids as templates to produce extended immobilised 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) hybridising the extended strands to the oligonucleotides comprising the second known region;

h) extending the second known region to form a duplex immobilised 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 immobilised extended strands comprising the first and second regions; and

g) amplifying the immobilised 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 pool of oligonucleotide probes comprises greater than 10³ different sequences.

10. The method of claim 9, wherein the pool of oligonucleotide probes 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. An array comprising a plurality of nucleic acid features, wherein each feature comprises:

i) a first nucleic acid sequence, comprising a first universal sequence common to all features and a first known region capable of hybridising to a first pre-selected region of a nucleic acid sequence of interest, and

ii) a second nucleic acid sequence, comprising a second universal sequence common to all features and a second known region capable of hybridising to a second pre-selected region of the nucleic acid sequence of interest.

15. The array according to claim 14 wherein the first and second known regions are not directly connected together.

16. The array of claim 14 wherein the first and second pre-selected regions in the sequence of interest are separated by less than 5000 base pairs.

17. The array of claim 16, wherein the first and second pre-selected regions in the sequence of interest are separated by between 10 base pairs and 2000 base pairs.

18. The array of claim 17, wherein the known first and second pre-selected regions in the sequence of interest are separated by between 50 base pairs and 500 base pairs.

19. The array of claim 14 wherein the number of features is at least 10⁶.

20. The array of claim 14 wherein the array is prepared by amplifying a probe library and cleaving the probe amplicons.

21. The array of claim 20 wherein the probe library is a pool of synthetic oligonucleotides.

22. A pool of oligonucleotides, each comprising universal sequences at both ends and two known regions that are capable of binding to two pre-selected regions of a nucleic acid sequence of interest, wherein the known regions are separated by a cleavage site.

23. The pool of claim 22, wherein the oligonucleotides are attached to a solid support.

24. The pool of claim 22, comprising greater than 10³ different sequences.

25. The pool of claim 22, wherein the two-preselected regions are separated by between 10 base pairs and 2000 base pairs in the sequence of interest.

26. A method of analysing a nucleic acid sample, the method comprising hybridising said nucleic acid sample to an array comprising a plurality of nucleic acid features, wherein each feature comprises a first universal sequence common to all features, a known region capable of hybridising to a pre-selected region of the nucleic acid sample of interest, and a second universal sequence common to all features.