Selection probe amplification

• US 20060183132A1
• Filed: 02/14/2005
• Published: 08/17/2006
• Est. Priority Date: 02/14/2005
• Status: Abandoned Application

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Perlegen Sciences, Inc.
• Inventors: Sparks, Andrew B., Ollmann, Amy, Sheehan, John, Stuve, Laura, Shen, Naiping, Fu, Glenn, Ballinger, Dennis
• Application Number:
  US11/058,432
• Publication Number:
  US 20060183132A1
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 2521/119 RNA polymerase
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2539/101 Subtraction analysis
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

Multiple unique selection probes are provided in a single medium. Each selection probe has a sequence that is complementary to a unique target sequence that may be present in a sample under consideration. For example, each selection probe may be complementary to a sequence that includes one of the SNPs used to genotype an organism. Single-stranded selection probes anneal or hybridize with sample sequences having the unique target sequences specified by the selection probe sequences. Sequences from the sample that do not anneal or hybridize with the selection probes are separated from the bound sequences by an appropriate technique. The bound sequences can then be freed to provide a mixture of isolated target sequences, which can be used as needed for the application at hand.

Claims

1. A method of isolating target nucleic acid sequences from a nucleic acid sample, the method comprising:

(a) generating nucleic acid fragments from the sample;

(b) amplifying the nucleic acid fragments;

(c) exposing the amplified nucleic acid fragments to at least about 2,000 distinct selection probes in a single reaction medium under conditions promoting annealing between the selection probes and the amplified nucleic acid fragments that are complementary to the selection probes, wherein the selection probes have sequences complementary to the target nucleic acid sequences;

(d) removing the amplified nucleic acid fragments that are not strongly bound to the selection probes; and

(e) releasing annealed amplified nucleic acid fragments from the selection probes, wherein said annealed amplified nucleic acid fragments are said target nucleic acid sequences, thereby isolating said target nucleic acid sequences.

2. The method of claim 1, further comprising characterizing the nucleic acid sample on the basis of the target nucleic acid sequences released in (e).

3. The method of claim 2, wherein the characterizing is performed by applying the target nucleic acid sequences to a nucleic acid array.

4. The method of claim 3, further comprising:

amplifying the target nucleic acid sequences released in (e); and

labelling said target nucleic acid sequences prior to contacting them with said nucleic acid array.

5. The method of claim 4, further comprising further fragmenting the target nucleic acid fragments prior to labelling.

6. The method of claim 1, wherein fragmenting the nucleic acid sample produces nucleic acid fragments having an average size of between about 25 and about 2,000 base pairs.

7. The method of claim 6 wherein the average size of the nucleic acid fragments is about 500 base pairs.

8. The method of claim 1, wherein generating nucleic acid fragments in (a) produces nucleic acid fragments having an average size that allows genotyping on a nucleic acid array without further fragmentation.

9. The method of claim 1, wherein amplifying the nucleic acid fragments comprises performing a Polymerase Chain Reaction (PCR) on substantially all of the nucleic acid fragments produced in (a).

10. The method of claim 1, further comprising, prior to amplifying the nucleic acid fragments, attaching adaptors to the ends of the nucleic acid fragments, wherein the adaptors comprise sequences complementary to primers employed in the amplification operation.

11. The method of claim 10, wherein the adaptors each comprise the same sequence.

12. The method of claim 10, wherein the adaptors comprise dsDNA with ssDNA tail.

13. The method of claim 10, wherein excess adaptors that do not attach to the ends of the nucleic acid fragments serve as primers in amplifying the nucleic acid fragments.

14. The method of claim 10, wherein attaching the adaptors comprises ligating the adaptors to blunt ends of the nucleic acid fragments.

15. The method of claim 1, wherein the selection probes comprise moieties that facilitate linkage to a solid substrate.

16. The method of claim 15, further comprising linking the selection probes to a solid substrate, wherein at least a subset of the selection probes is annealed to the amplified nucleic acid fragments between operations (c) and (d).

17. The method of claim 16, wherein the solid substrate comprises a plurality of beads.

18. The method of claim 16, wherein removing the amplified nucleic acid fragments that are not strongly bound to the selection probes comprises washing the solid substrate to remove unbound nucleic acid fragments.

19. The method of claim 18, wherein washing the solid substrate comprises exposing the solid substrate to a solution under conditions that remove partially annealed amplified nucleic acid fragments from bound selection probes.

20. The method of claim 1, wherein exposing the amplified nucleic acid fragments to the distinct selection probes in a single reaction medium, comprises providing at least about 50,000 distinct selection probes, each complementary to a distinct target nucleic acid sequence, in the single reaction medium.

21. The method of claim 20, wherein the number of distinct selection probes employed in the single reaction medium is between about 50,000 about 10⁷.

22. The method of claim 1, wherein exposing the amplified nucleic acid fragments to distinct selection probes in a single reaction medium comprises exposing the amplified nucleic acid fragments to at least about 5,000 distinct selection probes in said single reaction medium.

23. The method of claim 22, wherein exposing the amplified nucleic acid fragments to distinct selection probes in a single reaction medium comprises exposing the amplified nucleic acid fragments to at least about 10,000 distinct selection probes in said single reaction medium.

24. A method of isolating target nucleic acid fragments from a mixture of target and non-target nucleic acid fragments, the method comprising:

(a) applying an adaptor sequence to the ends of the target and non-target nucleic acid fragments in the mixture, wherein the adaptor sequence comprises a sequence between about 15 and 40 base pairs in length, and is present in excess to the number of nucleic acid fragment ends;

(b) performing a polymerase chain reaction to amplify the target and non-target fragments, wherein no primer sequence is necessary to amplify the target and non-target fragments besides that provided by denaturing excess adaptors;

(c) contacting the amplified target and non-target fragments with a plurality of selection probes simultaneously, under conditions that promote annealing of the selection probes and the target nucleic acid fragments, wherein the selection probes comprise sequences complementary to sequences of the target nucleic acid fragments; and

(d) separating the non-annealed and partially-annealed non-target nucleic acid fragments from the annealed target nucleic acid fragments, which are bound to said selection probes, thereby isolating the target nucleic acid fragments.

25. The method of claim 24, wherein the adaptor sequence is a double-stranded nucleic acid sequence.

26. The method of claim 25, wherein the adaptor has a blunt end for attachment to the ends of the nucleic acid fragments.

27. The method of claim 26, wherein the adaptor has a sticky end having an overhang that is not complementary to itself, whereby the sticky ends of the adaptor do not anneal to one another.

28. The method of claim 26, wherein one strand of the adaptor is lacking a moiety necessary for ligation at the blunt end of the adaptor, whereby the blunt ends of the adaptor do not ligate to one another.

29. The method of claim 24, wherein the adaptor is present in an excess of between about 10-100 fold over the number of nucleic acid fragment ends.

30. A set of selection probes for use in simultaneously selecting target nucleic acid fragments from non-target nucleic acid fragments, wherein the set comprises:

at least about 10,000 distinct selection probes in a common medium, each selection probe having a sequence complementary to a distinct target sequence including a distinct SNP, all found in a single genome,

wherein each of the distinct selection probes is between about 20 and 1000 base pairs in length.

31. The set of selection probes of claim 30, wherein the individual selection probes of the set are double-stranded nucleic acid sequences.

32. The set of selection probes of claim 30, wherein the set comprises between about 10⁴ and 10⁸ distinct selection probes.

33. The set of selection probes of claim 30, wherein the set comprises between about 10⁴ and 10⁵ distinct selection probes.

34. The set of selection probes of claim 30, wherein each of the distinct selection probes further comprises a moiety, apart from the selection probe sequence, that facilitates binding to a solid substrate.

35. The set of selection probes of claim 34, wherein the moiety is biotin or streptavidin.

36. The set of selection probes as recited in claim 30, wherein the individual selection probes of the set are prepared by PCR reactions specific for the individual selection probes.

37. A kit for isolating target nucleic acid fragments from non-target nucleic acid fragments, the kit comprising:

the set of selection probes as recited in claim 34;

and

a solid substrate comprising a surface feature for binding with the moiety on the selection probes and thereby facilitating immobilization of the selection probes on the substrate.

38. The kit of claim 37, further comprising primers and polymerase for amplifying the nucleic acid fragments.

39. The kit of claim 37, further comprising a nucleic acid array comprising sequences complementary to the target nucleic acid fragments.

40. The kit of claim 37, wherein the solid substrate comprises beads.