Complexity management of genomic DNA by locus specific amplification

• US 20050095645A1
• Filed: 12/23/2004
• Published: 05/05/2005
• Est. Priority Date: 06/17/2002
• Status: Abandoned Application

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Jones, Keith W., Shapero, Michael, Liu, Weiwei
• Application Number:
  US11/022,099
• Publication Number:
  US 20050095645A1
• Time in Patent Office: Days
• US Class Current: 435/6.14
• 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

1-25. -25. (canceled)

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.

46-78. -78. (canceled)

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.

82-85. -85. (canceled)