Complexity management of genomic DNA by locus specific amplification

• US 20030232348A1
• Filed: 10/14/2002
• Published: 12/18/2003
• Est. Priority Date: 06/17/2002
• Status: Active Grant

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Jones, Keith W., Shapero, Michael, Liu, Weiwei
• Granted Patent: US 7,108,976 B2
• Time in Patent Office: Days
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2521/307 Single strand endonuclease
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2531/113 PCR
• 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 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

The present invention provides for novel methods and kits for reducing the complexity of a nucleic acid sample to interrogate a collection of target sequences. In one embodiment complexity reduction can be accomplished by extension of a locus specific capture probe followed by amplification of the extended capture probe using common primers. The locus specific capture probes may be attached to a solid support. Multiple DNA sequences may be amplified simultaneously to produce a reduced complexity sample. The invention further provides for analysis of the above sample to interrogate sequences of interest such as polymorphisms. The amplified sample may be hybridized to an array, which may be specifically designed to interrogate the desired fragments for the presence or absence of a polymorphism.

Claims

1. A method of amplifying a collection of target sequences from a nucleic acid sample comprising:

generating a collection of capture probes comprising a plurality of different species of primers wherein each species comprises a first common sequence and a 3′

variable region that is specific for a target sequence in said collection of target sequences, wherein said collection of capture probes is attached to a solid support and the 3′

end of the capture probes is available for extension;

fragmenting the nucleic acid sample;

ligating an adapter to the fragments, said adapter comprising a second common sequence;

hybridizing the adapter-ligated fragments to the collection of capture probes;

extending the capture probes; and

amplifying the extended capture probes with first and second common sequence primers.

2. The method of claim 1 further comprising releasing the extended capture probes from the solid support prior to amplification.

3. The method of claim 2 wherein prior to releasing the extended capture probes from the solid support nucleic acids that are not covalently attached to the solid support are removed.

4. The method of claim 1 wherein said capture probes are attached to the solid support through a covalent interaction.

5. The method of claim 1 wherein the capture probes further comprise a tag sequence that is unique for each species of capture probe and the capture probes are attached to the solid support by hybridization to a collection of tag probes that are covalently attached to the solid support.

6. The method of claim 1 wherein each species of capture probe is attached to the solid support in a discrete location.

7. The method of claim 1 wherein prior to amplification the extended capture probes are enriched in the sample to be amplified.

8. The method of claim 1 wherein labeled nucleotides are incorporated into the extended capture probes and extended capture probes are isolated by affinity chromatography.

9. The method of claim 8 wherein said labeled nucleotides are labeled with biotin and avidin, streptavidin or an anti-biotin antibody is used to isolate extended capture probes.

10. The method of claim 1 wherein prior to amplification the extended capture probes are made double stranded and single stranded nucleic acid in the sample is digested.

11. The method of claim 10 wherein the single stranded nucleic acid in the sample is digested with a nuclease.

12. The method of claim 11 wherein the nuclease is Exonuclease I.

13. The method of claim 1 wherein prior to amplification the extended capture probes are circularized and uncircularized nucleic acid in the sample is digested.

14. The method of claim 13 wherein extended capture probes are circularized by a method comprising:

hybridizing an oligonucleotide splint to the extended capture probes, wherein the oligonucleotide splint is complementary to the first and second common sequences, thereby juxtaposing the 5′ and

3′

ends of extended capture probes; and

ligating the ends of the extended capture probes to form circular extended capture probes.

15. The method of claim 13 wherein the uncircularized nucleic acid remaining in the sample is digested with a nuclease.

16. The method of claim 15 wherein the nuclease is Exonuclease III.

17. The method of claim 1 wherein the nucleic acid sample is fragmented by digestion with one or more restriction enzymes.

18. The method of claim 1 wherein said capture probes are synthesized on a solid support.

19. The method of claim 1 wherein there are 100 to 1500 different target sequences in the collection of target sequences.

20. The method of claim 1 wherein there are 1,000 to 5,000 different target sequences in the collection of target sequences.

21. The method of claim 1 wherein there are 2,000 to 10,000 different target sequences in the collection of target sequences.

22. The method of claim 1 wherein there are 10,000 to 1,000,000 different target sequences in the collection of target sequences.

23. A method of analyzing a nucleic acid sample comprising:

amplifying a collection of target sequences from said nucleic acid sample according to the method of claim 1;

hybridizing the amplified collection of target sequences to an array; and

analyzing the hybridization pattern to detect the presence or absence of target sequences from the collection of target sequences.

24. A method of genotyping one or more polymorphic locations in a sample comprising:

amplifying a collection of target sequences from the sample according to the method of claim 1;

hybridizing the amplified collection of target sequences to an array designed to interrogate at least one polymorphic location in the collection of target sequences; and

analyzing the hybridization pattern to determine the identify of the allele or alleles present at one or more polymorphic location in the collection of target sequences.

25. A method for analyzing sequence variations in a population of individuals comprising;

obtaining a nucleic acid sample from each individual;

amplifying a collection of target sequences from each nucleic acid sample according to the method of claim 1;

hybridizing each amplified collection of target sequences to an array designed to interrogate sequence variation in the collection of target sequences to generate a hybridization pattern for each sample; and

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

26. A method of amplifying a collection of target sequences from a nucleic acid sample said method comprising:

generating a collection of capture probes comprising a plurality of different species of primers wherein each species comprises a first common sequence and a 3′

variable region that is specific for a target sequence in the collection of target sequences;

fragmenting the nucleic acid sample;

ligating an adapter to the fragments, wherein the adapter is ligated to the fragments so that the strand that is ligated to the 5′

end of the fragment strands comprises a second common sequence and the strand that is ligated to the 3′

end of the fragments lacks the complement of the second common sequence and is blocked from extension at the 3′

end;

hybridizing the adapter-ligated fragments to the collection of capture probes;

extending the capture probes; and

amplifying the extended capture probes with first and second common sequence primers.

27. The method of claim 26 wherein an amino group is used to block extension at the 3′

end of the adapter strand that is ligated to the 3′

end of the fragments.

28. A method of analyzing a nucleic acid sample comprising:

amplifying a collection of target sequences from said nucleic acid sample according to the method of claim 26;

hybridizing the amplified collection of target sequences to an array; and

analyzing the hybridization pattern to detect the presence or absence of target sequences from the collection of target sequences.

29. A method of genotyping one or more polymorphic locations in a sample comprising:

preparing an amplified collection of target sequences from the sample according to the method of claim 26;

hybridizing the amplified collection of target sequences to an array designed to interrogate at least one polymorphic location in the collection of target sequences; and

analyzing the hybridization pattern to determine the identify of the allele or alleles present at one or more polymorphic location in the collection of target sequences.

30. A method for analyzing sequence variations in a population of individuals comprising;

obtaining a nucleic acid sample from each individual;

amplifying a collection of target sequences from each nucleic acid sample according to the method of claim 26;

hybridizing each amplified collection of target sequences to an array designed to interrogate sequence variation in the collection of target sequences to generate a hybridization pattern for each sample; and

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

31. The method of claim 26 wherein the nucleic acid sample is fragmented by digestion with one or more restriction enzymes.

32. The method of claim 26 wherein prior to amplification the extension products are enriched in the sample to be amplified.

33. The method of claim 26 wherein labeled nucleotides are incorporated into the extension products and the extension products are enriched by affinity chromatography.

34. The method of claim 33 wherein said labeled nucleotides are labeled with biotin and avidin, streptavidin or an anti-biotin antibody is used to isolate extension products.

35. The method of claim 26 wherein prior to amplification the extended capture probes are made double stranded and single stranded nucleic acid in the sample is digested.

36. The method of claim 35 wherein the single stranded nucleic acid in the sample is digested with a nuclease.

37. The method of claim 36 wherein the nuclease is Exonuclease I.

38. The method of claim 26 wherein prior to amplification the extended capture probes are circularized and uncircularized nucleic acid in the sample is digested.

39. The method of claim 38 wherein extended capture probes are circularized by a method comprising:

hybridizing an oligonucleotide splint to the extended capture probes, wherein the splint is complementary to the first and second common sequences, thereby juxtaposing the 5′ and

3′

ends of extended capture probes; and

ligating the ends of the extended capture probes to form circular extended capture probes.

40. The method of claim 38 wherein the uncircularized nucleic acid remaining in the sample is digested with a nuclease.

41. The method of claim 40 wherein the nuclease is Exonuclease III.

42. The method of claim 26 wherein there are 100 to 1,500 different target sequences in the collection of target sequences.

43. The method of claim 26 wherein there are 1,000 to 5,000 different target sequences in the collection of target sequences.

44. The method of claim 26 wherein there are 2,000 to 10,000 different target sequences in the collection of target sequences.

45. The method of claim 26 wherein there are 10,000 to 1,000,000 different target sequences in the collection of target sequences.

46. A method for genotyping one or more polymorphisms in a nucleic acid sample comprising:

fragmenting the nucleic acid sample to generate fragments;

ligating an adaptor to the fragments said adapter comprising a first common sequence;

hybridizing a collection of capture probes to the fragments, wherein said capture probes comprise a second common sequence, a tag sequence unique for each species of capture probe, a first target specific sequence, a Type IIs restriction enzyme recognition sequence, and a second target specific sequence wherein the Type IIs restriction enzyme recognition sequence is positioned so that the enzyme will cut on the 5′

side of the polymorphic base;

extending said capture probes to generate extended capture probes;

amplifying the extended capture probes with first and second common sequence primers;

digesting the amplified product with a Type IIs restriction enzyme to generate amplified product fragments;

extending the amplified product fragments in at least one extension reaction;

hybridizing each extension reaction to an array comprising tag probes that hybridize to the tag sequences in the capture probes; and

analyzing the hybridization pattern on each of the arrays to determine at least one genotype.

47. The method of claim 46 wherein in each extension reaction there is at least one species of labeled ddNTP.

48. The method of claim 47 wherein one or more species of ddNTPs is labeled with biotin.

49. The method of claim 47 wherein there are four separate extension reactions wherein each extension reaction corresponds to a different species of labeled ddNTP and each extension reaction is hybridized to a different array.

50. The method of claim 47 wherein there are two separate extension reactions wherein two differentially labeled ddNTPs are present in each extension reaction and each extension reaction is hybridized to a different array.

51. The method of claim 47 wherein there is one extension reaction wherein four differentially labeled ddNTPs are present in the extension reaction and the extension reaction is hybridized to a single array.

52. The method of claim 46 wherein said capture probes are attached to a solid support.

53. The method of claim 52 wherein said capture probes are attached to the solid support through a covalent interaction.

54. The method of claim 52 wherein said capture probes are attached to said solid support by hybridization to a collection of tag probes that are attached to said solid support.

55. The method of claim 52 wherein each species of capture probe is attached to said solid support in a discrete location.

56. The method of claim 52 wherein said capture probes are synthesized on a solid support in a 5′

to 3′

direction.

57. The method of claim 46 wherein one of the common sequence primers is resistant to nuclease digestion and each extension reaction is digested with a 5′

to 3′

nuclease activity prior to hybridization to an array.

58. The method of claim 57 wherein the nuclease resistant common sequence primer comprises phosphorothioate linkages.

59. The method of claim 57 wherein said nuclease is T7 Gene 6 Exonuclease.

60. The method of claim 46 wherein prior to amplification the extended capture probes are enriched in the sample to be amplified.

61. The method of claim 46 wherein labeled nucleotides are incorporated into the extended capture probes and extended capture probes are isolated by affinity chromatography.

62. The method of claim 61 wherein said labeled nucleotides are labeled with biotin and avidin, streptavidin or an anti-biotin antibody is used to isolate extended capture probes.

63. The method of claim 46 wherein prior to amplification the extended capture probes are made double stranded and single stranded nucleic acid in the sample is digested.

64. The method of claim 63 wherein the single stranded nucleic acid in the sample is digested with a nuclease.

65. The method of claim 64 wherein the nuclease is Exonuclease I.

66. The method of claim 46 wherein prior to amplification the extended capture probes are circularized and uncircularized nucleic acid in the sample is digested.

67. The method of claim 66 wherein extended capture probes are circularized by a method comprising:

hybridizing an oligonucleotide splint to the extended capture probes, wherein the oligonucleotide splint is complementary to the first and second common sequences, thereby juxtaposing the 5′ and

3′

ends of extended capture probes; and

ligating the ends of the extended capture probes to form circular extended capture probes.

68. The method of claim 66 wherein the uncircularized nucleic acid remaining in the sample is digested with a nuclease.

69. The method of claim 68 wherein the nuclease is Exonuclease III.

70. The method of claim 46 wherein the nucleic acid sample is fragmented by digestion with one or more restriction enzymes.

71. The method of claim 46 wherein there are 100 to 1500 different target sequences in the collection of target sequences.

72. The method of claim 46 wherein there are 1,000 to 5,000 different target sequences in the collection of target sequences.

73. The method of claim 46 wherein there are 2,000 to 10,000 different target sequences in the collection of target sequences.

74. The method of claim 46 wherein there are 10,000 to 1,000,000 different target sequences in the collection of target sequences.

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

providing a nucleic acid sample from each individual;

genotyping each sample according to the method of claim 46;

and

comparing the genotypes from the samples to determine the presence or absence of sequence variation in the population of individuals.

76. A kit for amplifying a collection of target sequences said kit comprising:

a collection of capture probes, wherein each species of capture probe comprises a first common sequence and a 3′

variable region that is specific for a target sequence in said collection of target sequences;

an adapter comprising a second common sequence; and

a pair of first and second common sequence primers.

77. The kit of claim 76 wherein said collection of capture probes is covalently attached to a solid support and the 3′

end of the capture probes is available for extension.

78. The kit of claim 76 further comprising a restriction enzyme, buffer, DNA polymerase and dNTPs.

79. A kit for amplifying a collection of target sequences said kit comprising:

a collection of capture probes, wherein each species of capture probe comprises a first common sequence, a tag sequence unique for each species of capture probe, a first target specific sequence, a Type IIs restriction enzyme recognition sequence, and a second target specific sequence;

an adapter comprising a first strand comprising a second common sequence and a second strand that does not contain the complement of the second common sequence and is blocked from extension at the 3′

end; and

a pair of first and second common sequence primers.

80. The kit of claim 79 further comprising a Type IIs restriction enzyme, a ligase, dNTPs, ddNTPs, buffer and DNA polymerase.

81. The kit of claim 79 wherein one of the common sequence primers is resistant to nuclease digestion.

82. A collection of capture probes attached to a solid support, wherein said solid support is selected from the group consisting of arrays, beads, microparticles, microtitre dishes and gels.

83. A collection of capture probes, wherein each species of capture probe comprises a first common sequence, a tag sequence unique for each species of capture probe, a first target specific sequence, a Type IIs restriction enzyme recognition sequence, and a second target specific sequence, wherein the target specific sequence are specific for targets in a collection of target sequences.

84. A method of generating a plurality of oligonucleotides comprising:

synthesizing a plurality of oligonucleotides on a solid support and releasing the plurality of oligonucleotides from said solid support.

85. The method of claim 84 wherein said plurality of oligonucleotides comprises a collection of capture probes.