SELECTION PROBE AMPLIFICATION

• US 20090124514A1
• Filed: 10/24/2008
• Published: 05/14/2009
• Est. Priority Date: 02/26/2003
• Status: Abandoned Application

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Perlegen Sciences, Inc.
• Inventors: Sheehan, John, Pethiyagoda, Charit, Sparks, Andrew B., Ollmann, Amy, Stuve, Laura, Kennemer, Michael, Montgomery, Julie, Fu, Glenn, Ballinger, Dennis, Shen, Naiping
• Application Number:
  US12/258,244
• Publication Number:
  US 20090124514A1
• 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 2521/131 Terminal transferase
• 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 enriching a complex set of nucleic acids for a set of target nucleic acids, the method comprising:

(a) isolating the complex set of nucleic acids;

(b) amplifying the complex set of nucleic acids to produce amplified nucleic acids;

(c) exposing the amplified nucleic acids 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 acids that are complementary to the selection probes, wherein the selection probes have sequences complementary to the target nucleic acids;

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

(e) releasing annealed amplified nucleic acids from the selection probes, wherein said annealed amplified nucleic acids are said target nucleic acids, thereby enriching said complex set of nucleic acids for said set of target nucleic acids.

2. The method of claim 1, further comprising characterizing the complex set of nucleic acids on the basis of the target nucleic acids released in (e).

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

4. The method of claim 3, further comprising:

amplifying the target nucleic acids released in (e); and

labeling said target nucleic acids prior to contacting them with said nucleic acid array.

5. The method of claim 4, further comprising further fragmenting the target nucleic acids prior to said labeling.

6. The method of claim 2, wherein the characterizing is performed by sequencing the target nucleic acids.

7. The method of claim 6, wherein said sequencing is selected from the group consisting of pyrosequencing, deep sequencing, Sanger sequencing, SBS sequencing, and HANS sequencing.

8. The method of claim 6, wherein prior to step (b) the complex set of nucleic acids is subjected to denaturation and only one strand of each nucleic acid in the complex set of nucleic acids is subjected to said sequencing.

9. The method of claim 1, further comprising fragmenting said complex set of nucleic acids to produce nucleic acid fragments having an average size of between about 25 and about 2,000 base pairs.

10. The method of claim 9 wherein the average size of the nucleic acid fragments is about 800 base pairs.

11. The method of claim 9, wherein said average size of said nucleic acid fragments allows genotyping on a nucleic acid array without further fragmentation.

12. The method of claim 9, wherein amplifying the complex set of nucleic acids comprises performing a Polymerase Chain Reaction (PCR) on substantially all of the nucleic acid fragments.

13. The method of claim 1, further comprising attaching adaptors to the ends of the nucleic acids in the complex set of nucleic acids prior to said amplifying, wherein the adaptors comprise sequences complementary to primers employed in said amplifying.

14. The method of claim 13, wherein the adaptors each comprise the same sequence.

15. The method of claim 13, wherein the adaptors comprise dsDNA with ssDNA tail.

16. The method of claim 13, wherein excess adaptors that do not attach to the ends of the nucleic acids serve as primers in said amplifying.

17. The method of claim 13, wherein attaching the adaptors comprises ligating the adaptors to blunt ends of the nucleic acids in said complex set of nucleic acids.

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

19. The method of claim 18, further comprising linking the selection probes to a solid substrate, wherein at least a subset of the selection probes is annealed to at least a subset of the amplified nucleic acids during step (c).

20. The method of claim 19, wherein the solid substrate comprises a plurality of beads.

21. The method of claim 19, wherein removing the amplified nucleic acids that are not strongly bound to the selection probes comprises washing the solid substrate.

22. The method of claim 21, wherein washing the solid substrate comprises exposing the solid substrate to a solution under conditions that remove partially annealed amplified nucleic acids from bound selection probes.

23. The method of claim 1, wherein exposing the amplified nucleic acids 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.

24. The method of claim 23, wherein the number of distinct selection probes employed in the single reaction medium is between about 50,000 about 10⁷.

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

26. The method of claim 25, wherein exposing the amplified nucleic acids to distinct selection probes in a single reaction medium comprises exposing the amplified nucleic acids to at least about 10,000 distinct selection probes in said single reaction medium.

27. The method of claim 1, wherein a ratio said selection probes to said amplified nucleic acids present in the single reaction medium is at least 1:

1.

28. The method of claim 1, wherein a ratio of said selection probes to said amplified nucleic acids present in the single reaction medium is dependent on a complexity of said distinct selection probes and a complexity of said amplified nucleic acids.

29. The method of claim 28, wherein the ratio is about 1:

1 where the complexity of said distinct selection probes is about 10-30 Mb and the complexity of said amplified nucleic acids is about 3000 Mb.

30. The method of claim 28, wherein the ratio is about 1:

4 where the complexity of said distinct selection probes is about 3-5 Mb and the complexity of said amplified nucleic acids is about 3000 Mb.

31. The method of claim 28, wherein the ratio is about 1:

8 where the complexity of said distinct selection probes is about 0.5 Mb and the complexity of said amplified nucleic acids is about 3000 Mb.

32. The method of claim 1 further comprising performing a reselection by exposing the annealed amplified nucleic acids released in (e) to the selection probes as in step (c), removing those nucleic acids not strongly bound to the selection probes as in step (d), and releasing those annealed to the selection probes as in step (e), thereby further enriching said complex set of nucleic acids for said set of target nucleic acids.

33. The method of claim 1 further comprising performing an antiselection comprising:

(i) exposing the annealed amplified nucleic acids released in (e) to a set of antiselection probes in a single reaction medium under conditions promoting annealing between the selection probes and the amplified nucleic acids that are complementary to the antiselection probes, wherein the set of antiselection probes has sequences complementary to nucleic acids other than the target nucleic acids;

(ii) removing those nucleic acids strongly bound to the set of antiselection probes and retaining those nucleic acids not strongly bound to the set of antiselection probes, wherein said nucleic acids not strongly bound to the antiselection probes are target nucleic acids, thereby removing from said complex set of nucleic acids a set of nucleic acids that are not target nucleic acids and further enriching said complex set of nucleic acids for said set of target nucleic acids.

34. The method of claim 33 further comprising performing a reantiselection by reexposing the nucleic acids not strongly bound to the antiselection probes in (ii) to the set of antiselection probes as in step (i), removing those nucleic acids strongly bound to the antiselection probes and retaining those not strongly bound to the set of antiselection probes as in step (ii), thereby further removing from said complex set of nucleic acids a set of nucleic acids that are not target nucleic acids and further enriching said complex set of nucleic acids for said set of target nucleic acids.

35. The method of claim 1, wherein the at least about 2,000 distinct selection probes are complementary to only exonic sequences in the complex set of nucleic acids.

36. A method of enriching a complex set of nucleic acids for a set of target nucleic acids, the method comprising:

(a) amplifying the complex set of nucleic acids to produce amplified nucleic acids;

(b) exposing the amplified nucleic acids 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 acids that are complementary to the selection probes, wherein the selection probes have sequences complementary to the target nucleic acids;

(c) removing the amplified nucleic acids that are not strongly bound to the selection probes;

(d) releasing annealed amplified nucleic acids from the selection probes, wherein said annealed amplified nucleic acids are said target nucleic acids; and

(e) sequencing at least some of the target nucleic acids released in (d).

37. The method of claim 36, wherein said sequencing is selected from the group consisting of pyrosequencing, deep sequencing, Sanger sequencing, SBS sequencing, and HANS sequencing.

38. The method of claim 36, wherein said sequencing is pyrosequencing.

39. The method of claim 36, wherein prior to operation (a) the complex set of nucleic acids is subjected to denaturation and only one strand of each nucleic acid in the complex set of nucleic acids is subjected to said sequencing.

40. The method of claim 36, further comprising fragmenting said complex set of nucleic acids to produce nucleic acid fragments having an average size of between about 25 and about 2,000 base pairs.

41. The method of claim 36, further comprising attaching adaptors to the ends of the nucleic acids in the complex set of nucleic acids prior to said amplifying, wherein the adaptors comprise sequences complementary to primers employed in said amplifying.

42. The method of claim 41, wherein the adaptors each comprise the same sequence.

43. The method of claim 36, wherein the selection probes comprise moieties that facilitate linkage to a solid substrate.

44. The method of claim 43, further comprising linking the selection probes to a solid substrate, wherein at least a subset of the selection probes is annealed to at least a subset of the amplified nucleic acids during step (b).

45. The method of claim 36, wherein exposing the amplified nucleic acids 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.

46. The method of claim 36, wherein a ratio said selection probes to said amplified nucleic acids present in the single reaction medium is at least 1:

1.

47. The method of claim 36, further comprising performing a reselection by exposing the annealed amplified nucleic acids released in (d) to the selection probes as in step (b), removing those nucleic acids not strongly bound to the selection probes as in step (c), and releasing those annealed to the selection probes as in step (d), thereby further enriching said complex set of nucleic acids for said set of target nucleic acids.

48. The method of claim 36, wherein the at least about 2,000 distinct selection probes are complementary to only exonic sequences in the complex set of nucleic acids.

49. A method of enriching a complex set of nucleic acids for a set of target nucleic acids, the method comprising:

(a) amplifying the complex set of nucleic acids to produce amplified nucleic acids;

(b) exposing the amplified nucleic acids 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 acids that are complementary to the selection probes, wherein the selection probes have sequences complementary to the target nucleic acids;

(c) removing the amplified nucleic acids that are not strongly bound to the selection probes; and

(d) releasing annealed amplified nucleic acids from the selection probes, wherein said annealed amplified nucleic acids comprise less than about 3% of the sample.

50. The method of claim 49, further comprising fragmenting said complex set of nucleic acids to produce nucleic acid fragments having an average size of between about 25 and about 2,000 base pairs.

51. The method of claim 49, further comprising attaching adaptors to the ends of the nucleic acids in the complex set of nucleic acids prior to said amplifying, wherein the adaptors comprise sequences complementary to primers employed in said amplifying, and wherein the adaptors each comprise the same sequence.

52. The method of claim 49, wherein the selection probes comprise moieties that facilitate linkage to a solid substrate.

53. The method of claim 49, wherein exposing the amplified nucleic acids to the distinct selection probes in a single reaction medium, comprises providing at least about 10,000 distinct selection probes, each complementary to a distinct target nucleic acid sequence, in the single reaction medium.

54. The method of claim 49, wherein a ratio said selection probes to said amplified nucleic acids present in the single reaction medium is at least 1:

1.

55. The method of claim 49, further comprising performing a reselection by exposing the annealed amplified nucleic acids released in (d) to the selection probes as in step (b), removing those nucleic acids not strongly bound to the selection probes as in step (c), and releasing those annealed to the selection probes as in step (d), thereby further enriching said complex set of nucleic acids for said set of target nucleic acids.

56. The method of claim 49, wherein the at least about 2,000 distinct selection probes are complementary to only exonic sequences in the complex set of nucleic acids.

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

the set of selection probes comprising between about 10⁴ and 10⁵ distinct selection probes in a common medium, each selection probe having a sequence complementary to a distinct target sequence;

sequencing reagents comprising a sequencing primer, a DNA polymerase, ATP sulfurylase, luciferase, apyrase, adenosine 5′

-phosphosulfate, and luciferin; 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.