Methods for generating target specific probes for solution based capture

• US 8,986,958 B2
• Filed: 03/30/2010
• Issued: 03/24/2015
• Est. Priority Date: 03/30/2009
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Raymond, Christopher
• Primary Examiner(s): Wilder, Cynthia B
• Application Number:
  US13/260,906
• Publication Number:
  US 20120115744A1
• Time in Patent Office: 1,820 Days
• Field of Search: 435/91.2, 435/6.1
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 2525/151 repeat or repeated sequences, e.g. VNTR, microsatellite, concatemer
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2533/10 the purpose being to increase the length of an oligonucleotide strand

Abstract

Provided herein are compositions and kits for single-stranded nucleic acid probes, and methods for making the single-stranded nucleic acid probes, where the single-stranded nucleic acid probes comprise a probe region having a predetermined sequence which is flanked by a 5′ region having a first restriction enzyme recognition sequence and flanked by a 3′ region having a second restriction enzyme recognition sequence, and a region which hybridizes to a capture nucleic acid molecule. The single-stranded nucleic acid probes are useful for solution-based capture methods.

Claims

1. A method for generating a population of single-stranded nucleic acid probes, each probe comprising a predetermined nucleotide sequence, the method comprising:

a) providing a starting population of linear double-stranded nucleic acid precursor molecules each precursor molecule having (i) a probe region having the predetermined sequence which is flanked at a 5′ and

a 3′

end by a first and a second restriction enzyme recognition sequence for generating ligation substrates and for ligating a plurality of the double-stranded nucleic acid precursor molecules into head-to-tail concatemers (ii) the 5′

flanking region including the first restriction enzyme recognition sequence and (iii) the 3′

flanking region including the second restriction enzyme recognition sequence;

b) contacting the 5′ and

3′

flanking regions of the linear double-stranded nucleic acid precursor molecules with a first and a second restriction enzyme to cleave the first and second restriction enzyme recognition sequences so as to generate ligation substrates;

c) ligating the ligation substrates together so as to generate a plurality of random head-to-tail concatemers;

d) amplifying the plurality of head-to-tail concatemers;

e) contacting the amplified head-to-tail concatemers with the first and second restriction enzymes so as to release a plurality of double-stranded monomer linear precursor molecules; and

f) selectively removing one strand of the double-stranded monomer linear precursor molecules so as to generate a population of single-stranded nucleic acid probes, each probe comprising the predetermined nucleotide sequence.

2. The method of claim 1, wherein the single-stranded nucleic acid probes further comprise a region which hybridizes to a capture nucleic acid molecule.

3. The method of claim 1, wherein the members of the starting population of the linear double-stranded nucleic acid precursor molecules each comprise the same nucleotide sequence in the 5′

flanking region or each comprise the same nucleotide sequence in the 3′

flanking region.

4. The method of claim 1, wherein the 3′

flanking region further comprises a third restriction enzyme recognition sequence.

5. The method of claim 1, wherein the members of the starting population of the linear double-stranded nucleic acid precursor molecules each comprise the same predetermined sequences or different predetermined sequences.

6. The method of claim 1, wherein the ligation substrates of step (b) comprise overhanging nucleic acid ends capable of annealing together.

7. The method of claim 1, wherein the first or second restriction enzyme recognition sequence is cleaved by a type II restriction enzyme.

8. The method of claim 1, wherein the first or second restriction enzyme recognition sequence is cleaved by a Bsm1 enzyme.

9. The method of claim 1, wherein each predetermined nucleotide sequence in the population of linear double-stranded nucleic acid precursor molecules comprise a nucleotide sequence which is at least 95% identical to at least a portion of a sense or anti-sense strand of a target nucleic acid sequence.

10. The method of claim 9, wherein the predetermined sequence hybridizes to one target sequence or hybridizes to different target sequences.

11. The method of claim 9, wherein the predetermined sequences in the population of linear double-stranded nucleic acid precursor molecules hybridize to at least 10 different exon nucleotide sequences.

12. The method of claim 9, wherein the predetermined sequences in the population of linear double-stranded nucleic acid precursor molecules hybridize to at least 1000 different exon nucleotide sequences.

13. The method of claim 9, wherein the predetermined sequences hybridize to the target sequence at an interval of at least every 35 bases across the target sequence.

14. The method of claim 9, wherein the predetermined sequences hybridize to the target sequence of interest at an interval of one base across the target sequence.

15. The method of claim 1, wherein the probe region comprises 20-200 nucleotides.

16. The method of claim 1, wherein the predetermined nucleotide sequence comprises 10-50 nucleotides.

17. The method of claim 2, wherein the region of the single-stranded nucleic acid probe which hybridizes to the capture nucleic acid molecule comprises 10-50 nucleotides.

18. The method of claim 1, wherein the amplifying according to step (d) comprises isothermal amplification.

19. The method of claim 18, wherein the amplifying according to step (d) comprises random amplification primers.

20. The method of claim 19, wherein the random amplification primers each comprise a random 7-mer oligonucleotide and two additional nitroindole residues at the 5′

end.

21. The method of claim 19, wherein the random amplification primers each comprise a random 7-mer oligonucleotide and a phosphorothioate linkage to the 3′

end.

22. The method of claim 1, wherein the selectively removing one strand from the double-stranded monomer linear precursor molecules comprises:

a) contacting the released precursor molecules of step (e) with alkaline phosphatase;

b) contacting the released precursor molecules of step (e) with a third restriction enzyme which cleaves the third restriction enzyme recognition sequence; and

c) contacting the released precursor molecules of step (e) with an exonuclease so as to selectively degrade the one strand of the double-stranded monomer linear precursor molecules.

23. The method of claim 22, wherein the exonuclease is lambda exonuclease.

24. The method of claim 2, wherein the capture nucleic acid molecule further comprises a protein binding partner.

25. The method of claim 24, wherein the protein binding partner is biotin.

26. The method of claim 1, wherein each single-stranded nucleic acid probe comprises (i) the predetermined nucleotide sequence having a nucleotide sequence which is at least 95% identical to at least a portion of a sense or an anti-sense strand of a target nucleic acid sequence and (ii) a region which hybridizes to a capture nucleic acid molecule.

27. The method of claim 1, wherein the starting population of linear double-stranded nucleic acid precursor molecules is generated by steps comprising:

a) providing a population of a first single-stranded nucleic acid molecule comprising the 5′

flanking region, the probe region which comprises the predetermined sequence, and the capture sequence;

b) providing a population of a second single-stranded nucleic acid molecules comprising the sequence which is complementary to the capture sequence, and the 3′

flanking region;

c) annealing the first and second populations of the single-stranded nucleic acid molecules to form a nucleic acid duplex having overhanging 5′

ends; and

d) conducting a polymerase-dependent strand extension reaction on the overhanging 5′

ends so as to generate the population of double-stranded nucleic acid precursor molecules.

28. A method for enriching a target nucleic acid sequence of interest from a nucleic acid library, comprising:

a) contacting the population of single-stranded nucleic acid probes of claim 1 with the nucleic acid library having at least one target nucleic acid sequence of interest to form a mixture having unhybridized nucleic acid sequences and duplexes, each duplex having the single-stranded nucleic acid probe hybridized to the target nucleic acid sequence of interest;

b) contacting the duplexes with a population of capture nucleic acid molecules to form complexes having the single-stranded nucleic acid probe hybridized to the target nucleic acid sequence of interest and hybridized to the capture nucleic acid molecule;

c) separating the complex from the mixture; and

d) eluting the target nucleic acid sequence of interest from the complex.