Complexity Managment of genomic DNA

• US 20030036069A1
• Filed: 12/10/2001
• Published: 02/20/2003
• Est. Priority Date: 07/25/2001
• Status: Active Grant

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Su, Xing
• Granted Patent: US 6,872,529 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 2527/113 Time
• CPC Class Codes: C12Q 2527/143 Concentration of primer or probe

Abstract

The presently claimed invention provides for novel methods and kits for reducing the complexity of a nucleic acid sample by providing non-gel based methods for size fractionation. In a preferred embodiment, size fractionation can be accomplished by varying conditions or reagents of a PCR reaction to amplify fragments of specific size ranges. The invention further provides for analysis of the above sample by hybridization to an array, which may be specifically designed to interrogate the desired fragments for particular characteristics, such as, for example, the presence or absence of a polymorphism.

Claims

1. A method of reducing the complexity of a first nucleic acid sample to produce a second nucleic acid sample comprising:

fragmenting a first nucleic acid sample to produce fragments;

ligating one or more adaptors to the fragments;

digesting the fragments to produce single-stranded half molecules;

generating double-stranded fragments from the single-stranded half molecules;

amplifying a plurality of the fragments by a polymerase chain reaction (PCR), and

modulating the size of the amplified fragments by varying one or more reaction conditions or reagents to reduce the complexity of the first nucleic acid sample.

2. The method of claim 1 wherein the reaction condition or reagent varied is chosen from the group consisting of:

extension time, annealing time, primer concentration, primer length, presence or absence of 3′

to 5′

exonuclease activity, and concentration of nucleotide analogues.

3. The method of claim 1 wherein the adaptors are designed so that the 5′

and 3′

ends of the amplified fragments are complementary to one another.

4. The method of claim 3 wherein the complementarity is at least 10 bases long and is within 50 bases of the ends of the fragments.

5. The method of claim 3 wherein the complementarity is at least 10 bases long and is within 100 bases of the ends of the fragments.

6. The method of claim 1 where the reaction condition varied is the extension time of the PCR.

7. The method of claim 6 where the extension time is 2-5 seconds.

8. The method of claim 6 where the extension time is 5-10 seconds.

9. The method of claim 6 where the extension time is 10-30 seconds.

10. The method of claim 1 where the reaction condition varied is primer concentration.

11. The method of claim 10 where the primer concentration is 0.1-1 μ

M.

12. The method of claim 10 where the primer concentration is 0.1-10 μ

M.

13. The method of claim 10 where the primer concentration is 0.5 to 2.0 μ

M.

14. The method of claim 10 where the primer concentration is 0.3 to 5 μ

M.

15. The method of claim 1 where the reaction condition varied is primer length.

16. The method of claim 15 where the primer length is 10 to 100 bases.

17. The method of claim 15 where the primer length is 15 to 50 bases.

18. The method of claim 15 where the primer length is 20 to 35 bases.

19. The method of claim 1 where the reaction condition varied is the presence or absence of a 3′

to 5′

exonuclease activity.

20. The method of claim 1 where the reaction condition varied is the inclusion of one or more strand terminating nucleotides.

21. The method of claim 20 wherein the strand terminating nucleotides are selected from the following dideoxyribonucleotide triphosphates:

ddATP, ddTTP, ddGTP, ddCTP and ddUTP.

22. The method of claim 21 wherein the ratio of dNTP to ddNTP is 100 to 1.

23. The method of claim 21 wherein the ratio of dNTP to ddNTP is 1000 to 1.

24. The method of claim 1 further comprising the step of fractionating the fragments according to size by gel filtration chromatography prior to amplification.

25. The method of claim 1 wherein the step of fragmenting the first nucleic acid sample comprises digestion with at least one restriction enzyme.

26. The method of claim 1 wherein the step of fragmenting the first nucleic acid sample comprises digestion with a restriction enzyme that has a six base recognition sequence.

27. The method of claim 1 wherein the adaptor sequences comprise PCR primer template sequences.

28. The method of claim 1 wherein the second nucleic acid sample comprises at least 0.01% of the first nucleic acid sample.

29. The method of claim 1 wherein the second nucleic acid sample comprises at least 0.5% of the first nucleic acid sample.

30. The method of claim 1 wherein the second nucleic acid sample comprises at least 3% of the first nucleic acid sample.

31. The method of claim 1 wherein the second nucleic acid sample comprises at least 12% of the first nucleic acid sample.

32. The method of claim 1 wherein the second nucleic acid sample comprises at least 50% of the first nucleic acid sample.

33. The method of claim 1 wherein the first nucleic acid sample is genomic DNA, DNA, cDNA derived from RNA or mRNA.

34. The method of claim 1 wherein the size range of a substantial amount of the amplified DNA is 100 to 1000 base pairs.

35. The method of claim 1 wherein the size range of a substantial amount of the amplified DNA is 200 to 1200 base pairs.

36. The method of claim 1 wherein the size range of a substantial amount of the amplified DNA is 400 to 800 base pairs.

50. The method of claim 1 further comprising the steps of diluting the product of the PCR and amplifying the diluted product by a second round of PCR.

37. A method for analyzing a first nucleic acid sample comprising:

obtaining a second nucleic acid sample by;

fragmenting the first nucleic acid sample to produce fragments;

ligating one or more adaptors to the fragments; and

,

digesting the fragments to produce single stranded half molecules;

generating double stranded fragments from the single stranded half molecules;

amplifying a plurality of the fragments by a polymerase chain reaction (PCR) wherein the size of the amplified fragments is modulated by varying one or more reaction conditions or reagents to reduce the complexity of the first nucleic acid sample;

providing a nucleic acid array;

hybridizing the second nucleic acid sample to the array; and

analyzing a hybridization pattern resulting from the hybridization.

38. The method of claim 37 wherein the reaction condition or reagent varied is chosen from the group consisting of:

extension time, annealing time, primer concentration, primer length, presence or absence of 3′

to 5′

exonuclease activity, and concentration of nucleotide analogues.

39. The method of claim 37 wherein the method for analyzing a first nucleic acid sample comprises determining whether the first nucleic acid sample contains sequence variations.

40. The method of claim 39 wherein the sequence variations are single nucleotide polymorphisms (SNPs).

41. The method of claim 37 wherein the nucleic acid array is designed to query DNA fragments which have been produced by the procedures used to obtain the second nucleic acid sample.

42. The method of claim 37 wherein a substantial amount of the sequences predicted to be contained in the second nucleic acid sample are predetermined.

43. The method of claim 37 wherein a substantial amount of the sequences predicted to be contained in the second nucleic acid sample are first determined by a computer system.

44. A method of screening for DNA sequence variations in an individual comprising:

providing a first nucleic acid sample from the individual;

obtaining a second nucleic acid sample by;

fragmenting the first nucleic acid sample to produce fragments;

ligating adaptor sequences to the fragments;

digesting the fragments to produce single stranded half molecules;

generating double stranded fragments from the single stranded half molecules; and

,

amplifying a subset of the fragments by a polymerase chain reaction (PCR) wherein one or more reaction conditions or reagents are varied to favor amplification of a subset of fragments of a specific size range;

providing a nucleic acid array wherein the array comprises probes designed to interrogate for DNA sequence variations;

hybridizing the second nucleic acid sample to the array;

generating a hybridization pattern resulting from the hybridization; and

determining the presence or absence of DNA sequence variations in the individual based upon an analysis of the hybridization pattern.

45. The method of claim 44 wherein the sequence variation is a single nucleotide polymorphism (SNP).

46. The method of claim 44 wherein the SNP is associated with a disease.

47. The method of claim 44 wherein the SNP is associated with the efficacy of a drug.

48. A method for screening for DNA sequence variations in a population of individuals comprising:

providing a first nucleic acid sample from each of the individuals;

providing a second nucleic acid sample by;

fragmenting the first nucleic acid sample to produce fragments;

ligating adaptor sequences to the fragments;

digesting the fragments to produce single stranded half molecules;

generating double stranded fragments from the single stranded half molecules;

amplifying a subset of the fragments by a polymerase chain reaction (PCR) wherein one or more reaction conditions or reagents are varied to favor amplification of a subset of fragments of a specific size range;

providing a plurality of nucleic acid arrays wherein the arrays comprise probes designed to interrogate for DNA sequence variations;

hybridizing each of the second nucleic acid samples to one of the plurality of arrays;

generating a plurality of hybridization patterns resulting from the hybridizations; and

analyzing the hybridization patterns to determine the presence or absence of sequence variation in the population of individuals.

49. The method of claim 48 wherein the sequence variation is a single nucleotide polymorphism (SNP).

51. A method of reducing the complexity of a first nucleic acid sample to produce a second nucleic acid sample whereby the second nucleic acid sample is obtainable by:

fragmenting the first nucleic acid sample to produce fragments;

ligating adaptor sequences to both ends of the fragments such that the 5′ and

3′

ends of the fragments are complementary to one another;

digesting the fragments to produce single stranded half molecules;

generating double stranded fragments from the single stranded half molecules; and

,

amplifying a subset of the fragments by a polymerase chain reaction (PCR) wherein a subset of fragments of a specific size range are preferentially amplified by varying the PCR primer concentration.

52. The method of claim 51 where the primer concentration is 0.1-1 μ

M.

53. The method of claim 51 where the primer concentration is 0.1-10 μ

M.

54. The method of claim 51 where the primer concentration is 0.5 to 2.0 μ

M.

55. The method of claim 51 where the primer concentration is 0.3 to 0.5 μ

M.

56. A method of genotyping an individual comprising:

identifying a collection of SNPs that are found on fragments of a selected size range resulting from digestion with one or more selected restriction enzymes;

designing an array to interrogate the collection of SNPs;

providing a first nucleic acid sample from the individual;

fragmenting the first nucleic acid sample with the one or more selected restriction enzymes;

digesting the fragments to produce single stranded half molecules;

generating double stranded fragments from the single stranded half molecules;

amplifying the fragments by PCR wherein a subset of fragments of the selected size range are preferentially amplified;

hybridizing the PCR product to an array; and

analyzing the hybridization pattern to determine the presence or absence of the collection of SNPs.

57. A kit for reducing the complexity of a nucleic acid sample comprising:

buffer, nucleotide triphosphates, one or more reverse transcriptases, one or more nucleases, one or more restriction enzymes, one or more adaptors, one or more ligases, one or more DNA polymerases, one or more primers and instructions for the use of the kit.