Methods of using nick translate libraries for snp analysis

• US 20040197791A1
• Filed: 12/18/2003
• Published: 10/07/2004
• Est. Priority Date: 06/29/2001
• Status: Abandoned Application

• Current Assignee: Rubicon Genomics, Inc
• Original Assignee: Rubicon Genomics, Inc
• Inventors: Makarov, Vladimir L., Langmore, John P.
• Application Number:
  US10/481,488
• Publication Number:
  US 20040197791A1
• Time in Patent Office: Days
• US Class Current: 435/6.14
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2521/307 Single strand endonuclease
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2533/101 Primer extension
• 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

Abstract

The present invention is directed to amplification of a single nucleotide polymorphism by utilizing a library of nick translate molecules. The methods are also directed to highly multiplexed amplification of a nucleic acid sequence to facilitate detection of a single nucleotide polymorphism.

Claims

1. A method of amplifying a single nucleotide polymorphism (SNP) from a DNA sample, comprising:

a) obtaining the DNA sample comprising said single nucleotide polymorphism to be amplified;

b) generating at least one nick translate molecule from said DNA sample, wherein said nick translate molecule comprises said single nucleotide polymorphism; and

c) amplifying said nick translate molecule.

2. The method of claim 1, wherein said step of generating the nick translate molecule comprises:

a) attaching upstream adaptor molecules to ends of DNA sample molecules to provide a nick translation initiation site;

b) subjecting the DNA molecules to nick translation comprising DNA polymerization and 5′

-3′

exonuclease activity to produce the nick translate molecules; and

c) attaching downstream adaptor molecules to the nick translate molecules to produce adaptor attached nick translate molecules.

3. A method of producing a library of SNP-containing DNA molecules, comprising:

a) obtaining a DNA sample comprising at least one SNP;

b) digesting DNA molecules of the DNA sample with a sequence-specific endonuclease;

c) attaching upstream adaptor molecules to ends of DNA molecules of the sample to provide a nick translation initiation site;

d) subjecting the DNA molecules to nick translation comprising DNA polymerization and 5′

-3′

exonuclease activity to produce the nick translate molecules, wherein said nick translate molecules comprise said SNP;

e) attaching downstream adaptor molecules to the nick translate molecules to produce adaptor attached nick translate molecules; and

f) separating the SNP-containing nick translate molecules.

4. The method of claim 3, wherein said separating step is by size.

5. The method of claim 3, wherein said separating step is by hybridization.

6. The method of claim 3, wherein said separating step further comprises amplification of at least one said SNP-containing nick translate molecules.

7. The method of claim 6, wherein said amplification is by polymerase chain reaction.

8. A method of analyzing a SNP from a plurality of DNA samples, comprising:

a) obtaining said plurality of DNA samples, wherein at least one DNA sample comprises said SNP;

b) digesting DNA molecules of the DNA sample with a sequence-specific endonuclease;

c) attaching upstream adaptor molecules to ends of DNA molecules of the sample to provide a nick translation initiation site;

d) subjecting the DNA molecules to nick translation comprising DNA polymerization and 5′

-3′

exonuclease activity to produce the nick translate molecules;

wherein said nick translate molecules comprise said at least one SNP;

e) attaching downstream adaptor molecules to the nick translate molecules to produce adaptor attached nick translate molecules; and

f) separating the SNP-containing nick translate molecules.

9. The method of claim 8, wherein the upstream adaptors are nonidentical.

10. The method of claim 8, wherein said separating step is by size.

11. The method of claim 8, wherein said separating step is by hybridization.

12. The method of claim 8, wherein said separating step further comprises amplification of said SNP-containing nick translate molecules.

13. A method of isolating a specific SNP-containing nick translate molecule from a plurality of nick translate molecules, comprising:

a) obtaining a plurality of SNP-containing nick translate molecules;

b) ligating to an end of the SNP-containing nick translate molecules a first oligonucleotide to form a first oligonucleotide-nick translate molecule complex, wherein said first oligonucleotide comprises

i) nucleic acid sequence complementary to an adaptor end of said nick translate molecules;

ii) a double stranded region;

wherein the double stranded region facilitates the formation of an adjacent hairpin or loop in the oligonucleotide;

iii) a free 3′

OH; and

iv) a 5′

phosphate;

c) attaching to said first oligonucleotide-nick translate molecule complex a second oligonucleotide to form a first oligonucleotide-nick translate molecule-second oligonucleotide-complex, wherein the second oligonucleotide comprises;

i) nucleic acid sequence adjacent to an adaptor end of said nick translate molecules;

ii) nucleic acid sequence nonidentical to a restriction endonuclease site used in generating the nick translate molecules; and

iii) an affinity tag;

d) isolating the nick translate molecule-first oligonucleotide-second oligonucleotide-complex from said plurality of nick translate molecules by said affinity tag.

14. The method of claim 13, wherein said attaching step further comprises ligation of said second oligonucleotide to said first oligonucleotide-nick translate molecule complex.

15. The method of claim 13, wherein said first oligonucleotide further comprises a labile base.

16. The method of claim 13, wherein said double stranded region of said first oligonucleotide is approximately six to eight bases.

17. The method of claim 13, wherein said double stranded region of said first oligonucleotide is at least about 4 bases.

18. The method of claim 13, wherein said double stranded region of said first oligonucleotide is no more than about 100 bases.

19. The method of claim 13, wherein said nucleic acid sequence in said second oligonucleotide which corresponds to the nucleic acid sequence adjacent to an adaptor end of said nick translate molecules is five nucleotides in length.

20. The method of claim 13, wherein the affinity tag of said second oligonucleotide is biotin.

21. A method of isolating a complementary nucleic acid molecule to a specific SNP-containing nick translate molecule, comprising:

a) obtaining a plurality of nick translate molecules;

b) introducing to said plurality an oligonucleotide comprising;

i) a nucleic acid sequence complementary to a specific region of said specific nick translate molecule;

ii) a nucleic acid sequence substantially nonidentical to a sequence in said specific nick translate molecule, wherein the nucleic acid sequence is 5′

to said sequence in i); and

iii) an affinity tag,

wherein the oligonucleotide hybridizes to the specific nick translate molecule;

c) extending the oligonucleotide by polymerization to form a complementary nucleic acid molecule for the specific nick translate molecule; and

d) isolating the extended complementary nucleic acid sequence molecule from the plurality of nick translate molecules.

22. The method of claim 21, wherein the method further comprises amplifying said complementary nucleic acid molecule.

23. The method of claim 22, wherein said amplification step is by polymerase chain reaction.

24. The method of claim 21, wherein the oligonucleotide further comprises a hairpin or loop structure.

25. A method of amplifying a nucleic acid sequence for SNP analysis, comprising:

a) generating a nick translate molecule comprising the nucleic acid sequence and comprising an upstream adaptor and a downstream adaptor;

b) performing polymerase chain reaction to amplify said nick translate molecule using a first oligonucleotide complementary to an adaptor sequence of said nick translate molecule and a second oligonucleotide complementary to a known nucleic acid sequence of said nick translate molecule.

26. The method of claim 25, wherein the step of generating said nick translate molecule comprises:

a) attaching said upstream adaptor molecule to ends of DNA molecules comprising said nucleic acid sequence for SNP analysis to provide a nick translation initiation site;

b) subjecting the DNA molecules to nick translation comprising DNA polymerization and 5′

-3′

exonuclease activity to produce the nick translate molecules; and

c) attaching downstream adaptor molecules to the nick translate molecules to produce adaptor attached nick translate molecules.

27. A method of multiplex amplification of a plurality of nucleic acid sequences for SNP analysis, comprising:

a) generating a plurality of nick translate molecules comprising a nucleic acid sequence comprising said SNP, wherein each nick translate molecule comprises a first adaptor and a second adaptor;

b) introducing to said plurality of nick translate molecules a plurality of first oligonucleotides complementary to said first or second adaptor sequence of said nick translate molecules and a plurality of second oligonucleotides, wherein each second oligonucleotide is complementary to a known nucleic acid sequence in a nick translate molecule; and

c) amplifying the region in the nucleic acid sequence of said nick translate molecules between said first oligonucleotide and said second oligonucleotide by polymerase chain reaction.

28. A method of multiplex amplification of a plurality of nucleic acid sequences for SNP analysis, comprising:

a) generating a plurality of nick translate molecules each comprising a nucleic acid sequence comprising said SNP, wherein each nick translate molecule comprises a first adaptor and a second adaptor;

b) introducing to said plurality of nick translate molecules a plurality of first oligonucleotides complementary to said first adaptor sequence of said nick translate molecules and a plurality of second oligonucleotides, wherein the second oligonucleotide comprises

i) nucleic acid sequence complementary to said second adaptor; and

ii) multiple nucleotide bases at the 3 terminal end of said second oligonucleotide which are complementary to corresponding multiple nucleotide bases in the nucleic acid sequence of said nick translate molecule immediately adjacent to said second adaptor;

c) amplifying the region in the nucleic acid sequence of said nick translate molecules between said first oligonucleotide and said second oligonucleotide by polymerase chain reaction, whereby the amplification of the nucleic acid sequence occurs only under conditions wherein the second oligonucleotide anneals to said nick translate molecule at said multiple nucleotide bases immediately adjacent to the second adaptor.

29. The method of claim 28, wherein said multiple nucleotide bases comprise two bases.

30. The method of claim 28, wherein said multiple nucleotide bases comprise three bases.

31. A method of multiplex amplification of a nucleic acid sequence comprising a SNP of interest, wherein the nucleic acid sequence is adjacent to a known nucleic acid sequence, comprising:

a) obtaining a DNA sample;

b) processing said DNA sample to generate a library of nick translate molecules, wherein said nick translate molecules are separated into sublibraries of molecules that are complementary to specified positions within a region of the DNA, and wherein said sublibraries are partitioned into chambers of a solid support; and

c) amplifying by polymerase chain reaction within said chambers at least one nick translate molecule or fragment thereof using a primer from said known nucleic acid sequence.

32. The method of claim 31, wherein said DNA sample further comprises a genome.

33. The method of claim 31, wherein said solid support is a microwell plate.

34. A method of multiplex amplification of a nucleic acid sequence comprising a SNP of interest, wherein the nucleic acid sequence is adjacent to a known nucleic acid sequence, comprising:

a) obtaining a DNA sample;

b) processing said DNA sample to generate a library of nick translate molecules, wherein said nick translate molecules are in a pooled collection and wherein the nick translate molecules are comprised of sequences complementary to unknown positions within a region of the template DNA; and

c) amplifying by polymerase chain reaction within said pooled collection at least one nick translate molecule or fragment thereof using a primer from said known nucleic acid sequence.

35. The method of claim 34, wherein said pooled collection is in a single tube.

36. The method of claim 34, further comprising applying said amplified nick translate molecules to a DNA microarray, wherein hybridization of a nick translate molecule to the DNA microarray identifies said SNP.

37. A method of assaying a DNA sample for the presence of multiple specific SNPs, comprising:

a) generating a plurality of nick translate molecules from said DNA molecules of said sample, wherein said plurality of nick translate molecules comprise said multiple SNPs;

b) introducing to said nick translate molecules a plurality of oligonucleotides, wherein an oligonucleotide hybridizes adjacent to a specific SNP location and wherein the 3′

base of said oligonucleotide is variable;

c) extending by polymerization from said oligonucleotide, whereby extension only occurs if said variable 3′

base of said oligonucleotide is complementary to the corresponding nucleotide of said specific SNP; and

d) detecting said extended oligonucleotide.

38. The method of claim 37, wherein said detection step further comprises separation by size.

39. The method of claim 38, wherein said size detection is by capillary electrophoresis.

40. The method of claim 37, wherein said extended oligonucleotide is detected by detecting a label on the 3′

base of said oligonucleotide.

41. The method of claim 40, wherein said label is fluorescent.

42. The method of claim 37, wherein multiple specific SNPs are detected concomitantly, and wherein the labels for multiple nonidentical oligonucleotides in said plurality of oligonucleotides are distinguishable.

43. A method of assaying a DNA sample for the presence of multiple specific SNPs, comprising:

a) generating a plurality of nick translate molecules from said DNA molecules of said sample, wherein said plurality of nick translate molecules comprise said SNP;

b) introducing to said nick translate molecules a plurality of first oligonucleotides, wherein a first oligonucleotide hybridizes such that its 5′

end is adjacent to a specific SNP;

c) extending said first oligonucleotide by primer extension to form a plurality of nick translate molecule-first oligonucleotide extension product hybrids;

d) introducing to said plurality of hybrids a plurality of second oligonucleotides, wherein a second oligonucleotide hybridizes adjacent to the specific SNP and comprises a variable nucleotide 3′

end; and

e) ligating the 3′

end of said second oligonucleotide to the 5′

end of said first oligonucleotide extension product, whereby said ligation occurs only if said variable nucleotide is complementary to said SNP, to form a ligated molecule of said second oligonucleotide and said first oligonucleotide extension product; and

f) detecting said ligated molecule.

44. The method of claim 43, wherein said second oligonucleotide is fluorescently labeled.

45. The method of claim 43, wherein said plurality of second oligonucleotides are differentially fluorescently labeled.

46. The method of claim 43, wherein said detection step of said ligated molecule further comprises separation by size.

47. The method of claim 46, wherein said size separation is by capillary electrophoresis.

48. A method of analyzing at least one SNP from a plurality of individuals, comprising:

a) generating at least one specific nick translate molecule from DNA samples from each individual, wherein said specific nick translate molecule comprises the SNP; and

b) detecting said SNP.

49. The method of claim 48, wherein said detection step further comprises:

a) introducing to the nick translate molecule from the plurality of individuals a plurality of oligonucleotides, wherein said oligonucleotides hybridize adjacent to said SNP and wherein the 3′

base of said oligonucleotide is variable;

b) extending by polymerization from said oligonucleotide, whereby extension only occurs if said variable 3′

base of said oligonucleotide is complementary to the corresponding nucleotide of said SNP; and

c) detecting said extended oligonucleotide.

50. The method of claim 49, wherein said method further comprises separating said extended oligonucleotides by size.

51. The method of claim 50, wherein said size separation is by electrophoresis.

52. The method of claim 49, wherein said extended oligonucleotides are detected by fluorescent label.

53. The method of claim 48, wherein said detection step further comprises:

a) introducing to the nick translate molecules from the plurality of individuals a plurality of first oligonucleotides, wherein a first oligonucleotide hybridizes such that its 5′

end is adjacent to the SNP;

b) extending said first oligonucleotide by primer extension to form a plurality of nick translate molecule-first oligonucleotide extension product hybrids;

c) introducing to said plurality of hybrids a plurality of second oligonucleotides, wherein a second oligonucleotide hybridizes adjacent to the SNP and comprises a variable nucleotide 3′

end; and

d) ligating the 3′

end of said second oligonucleotide to the 5′

end of said first oligonucleotide extension product, whereby said ligation occurs only if said variable nucleotide is complementary to said SNP, to form a ligated molecule of said second oligonucleotide and said first oligonucleotide extension product; and

e) detecting said ligated molecule.

54. The method of claim 53, wherein said detection step further comprises separating said ligated molecules by size.

55. The method of claim 54, wherein said size separation is by electrophoresis.

56. The method of claim 54, wherein said extended oligonucleotides are detected by fluorescent label.

57. A method of analyzing at least one SNP from DNA samples from a plurality of individuals, comprising:

a) generating from each of said DNA samples a specific nick translate molecule comprising said SNP, wherein an adaptor on one end of said nick translate molecule comprises a unique nucleic acid sequence;

b) introducing to said nick translate molecules a two-part oligonucleotide, comprising;

i) a first part comprising nucleic acid sequence complementary to the unique nucleic acid sequence of said adaptor; and

ii) a second part comprising nucleic acid sequence complementary to nucleic acid sequence immediately 5′

to the SNP;

whereby said introduction results in the hybridization of said two parts of the oligonucleotide to the respective complementary sequences of said nick translate molecule and results in the formation of a loop in said nick translate molecule to bring said two parts in proximity of each other;

c) introducing to said two-part oligonucleotide differentially fluorescently labeled dideoxynucleotide triphosphates and DNA polymerase;

d) incorporating into the two-part oligonucleotide the fluorescently labeled dideoxynucleotide triphosphate which is complementary to said SNP; and

e) detecting said SNP.

58. The method of claim 57, wherein said SNP detection step further comprises hybridization of said fluorescently labeled dideoxynucleotide triphosphate-incorporated two-part oligonucleotide to a solid support, wherein the solid support comprises multiple positions, wherein each position comprises a unique adaptor sequence.

59. The method of claim 58, wherein said solid support is a chip.

60. A method of amplification of a genome comprising a SNP of interest, comprising:

a) obtaining the genome;

b) generating a plurality of nick translate molecules from said genome, wherein at least one nick translate molecule comprises the SNP of interest; and

c) amplifying the SNP-containing nick translate molecule.

61. The method of claim 60, further comprising detection of said SNP.

62. The method of claim 61, wherein said SNP is detected by microarray analysis, sequencing, hybridization, or a combination thereof.

63. The method of claim 60, wherein said generating of the nick translate molecules comprises:

a) attaching upstream adaptor molecules to ends of DNA molecules in the genome to provide a nick translation initiation site;

b) subjecting the DNA molecules to nick translation comprising DNA polymerization and 5′

-3′

exonuclease activity to produce the nick translate molecules; and

c) attaching downstream adaptor molecules to the nick translate molecules to produce adaptor attached nick translate molecules.