Methods, compositions, and kits for detecting allelic variants

• US 9,534,255 B2
• Filed: 12/17/2009
• Issued: 01/03/2017
• Est. Priority Date: 12/17/2008
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Chen, Caifu, Tan, Ruoying
• Primary Examiner(s): THOMAS, DAVID C
• Application Number:
  US12/641,321
• Publication Number:
  US 20100221717A1
• Time in Patent Office: 2,574 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2537/161 A competitive reaction step
• CPC Class Codes: C12Q 2561/101 Taqman
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/172 Haplotypes

Abstract

In some embodiments, the present inventions relates generally to compositions, methods and kits for use in discriminating sequence variation between different alleles. More specifically, in some embodiments, the present invention provides for compositions, methods and kits for quantitating rare (e.g., mutant) allelic variants, such as SNPs, or nucleotide (NT) insertions or deletions, in samples comprising abundant (e.g., wild type) allelic variants with high specificity and selectivity. In particular, in some embodiments, the invention relates to a highly selective method for mutation detection referred to as competitive allele-specific TaqMan PCR (“cast-PCR”).

Claims

1. A composition comprising:

a) an allele-specific primer that is complementary to a target sequence comprising a first allelic variant;

b) an allele-specific blocker probe that does not comprise a label and is complementary to said target sequence comprising a second allelic variant, wherein said second allelic variant corresponds to a position in said target sequence that is at the same loci as said first allelic variant in said target sequence, and wherein said allele-specific blocker probe comprises a minor groove binder (MGB) at the 3′

end that is non-extendable;

c) a locus-specific primer that is complementary to a region of the target sequence that is 3′

from the first allelic variant and on the opposite strand; and

d) a locus-specific detector probe wherein the detector probe is a 5′

nuclease probe and has two labels.

2. The composition of claim 1, wherein the two labels of the locus-specific detector probe comprise a fluorophore and a quencher.

3. The composition of claim 1, wherein said allele-specific primer comprises a tail.

4. The composition of claim 3, wherein said tail is GC-rich.

5. The composition of claim 3, wherein said tail is between 2-30 nucleotides long.

6. The composition of claim 3, wherein said tail is at the 5′

end of said allele-specific primer.

7. The composition of claim 1, wherein said allele-specific primer has a melting temperature (Tm) between 50°

C. to 55°

C.

8. The composition of claim 1, wherein said allele-specific primer is at a concentration between 20-900 nM.

9. The composition of claim 1, wherein said allele-specific primer is designed to comprise a highly discriminating base at the 3′

terminus.

10. The composition of claim 1, wherein the 3′

-most nucleotide of said allele-specific primer is complementary to said first allelic variant.

11. The composition of claim 10, wherein said 3′

-most nucleotide is A if T is said first allelic variant;

or G the if C is said first allelic variant.

12. The composition of claim 1, wherein said allele-specific primer and/or said allele-specific blocker probe comprises at least one modified base.

13. The composition of claim 12, wherein said modified base is a 8-aza-7-deaza-dN (ppN) base analog, where N is adenine (A), cytosine (C), guanine (G), or thymine (T).

14. The composition of claim 12, wherein said modified base is a locked nucleic acid (LNA) base.

15. The composition of claim 12, wherein said modified base is any modified base that increases the Δ

Tm between matched and mismatched target sequences.

16. The composition of claim 1, wherein said allele-specific blocker probe further comprises an MGB moiety at the 5′

-end and/or at an internal position within said allele-specific blocker probe.

17. The composition of claim 16, wherein said allele-specific blocker probe comprises a 3′

end that is non-extendable.

18. The composition of claim 1, wherein said locus-specific detector probe further comprises a minor groove binder.

19. The composition of claim 1, wherein at least one of the two labels comprises a reporter moiety.

20. The composition of claim 2 further comprising a minor grove binder covalently attached to the quencher in said locus-specific detector probe.

21. The composition of claim 1, wherein said composition further comprises a nucleic acid molecule, a polymerase, dNTPs, and/or other reagents or buffers.

22. The composition of claim 21, wherein said nucleic acid molecule is genomic DNA (gDNA).

23. The composition of claim 22, wherein said polymerase is thermostable.

24. The composition of claim 21, wherein said nucleic acid molecule is derived from a tumor sample.

25. The composition of claim 21, wherein said nucleic acid molecule is derived from a blood sample comprising circulating tumor cells.

26. The composition of claim 24, wherein said tumor sample is a breast or a lung cancer tumor sample.

27. The composition of claim 24, wherein said tumor comprises mutations in Ras, EGFR, Kit, pTEN, and/or p53.

28. The composition of claim 27, wherein said Ras mutation is a KRAS or an NRAS mutation.

29. The composition of claim 28, wherein said KRAS mutation is in codon 12 and/or codon 13 as depicted in FIG. 6.

55. The composition of claim 1, further comprising a polymerase with 5′

to 3′

exonuclease activity.

56. The composition of claim 55, wherein said polymerase is Taq polymerase.

57. The composition of claim 1, wherein said allele-specific blocker probe is at a concentration that is less than the concentration of said allele-specific primer.

58. The composition of claim 1, wherein said second allelic variant is located 7-15 nucleotides away from said minor groove binder when said allele-specific blocker probe is hybridized to said target sequence.

59. The composition of claim 1, wherein said allele-specific blocker probe does not comprise a base analog.

60. The composition of claim 1, wherein said allele-specific primer and said allele-specific blocker probe both comprise one or more base analogs.

61. The composition of claim 1, wherein said allele-specific blocker probe has a Tm that is between 60°

C. to 66°

C. and said allele-specific primer has a Tm that is between 50°

C. to 70°

C.

30. A kit comprising, two or more containers comprising the following components independently distributed in one of the two or more containers:

a) an allele-specific primer that is complementary to a target sequence comprising a first allelic variant; and

b) an allele-specific blocker probe that does not comprise a label and is complementary to said target sequence comprising a second allelic variant, wherein said second allelic variant corresponds to a position in said target sequence that is the same as said first allelic variant in said target sequence, and wherein said allele-specific blocker probe comprises a minor groove binder (MGB) at the 3′

end that is non-extendable;

c) a locus-specific primer that is complementary to a region of said target sequence that is 3′

from said first allelic variant and on the opposite strand; and

d) a locus-specific detector probe wherein the detector probe is a 5′

nuclease probe and wherein the detector probe has two labels.

31. The kit of claim 30, wherein the two labels of the locus-specific detector probe comprise a fluorophore and a quencher.

32. The kit of claim 30, wherein said allele-specific primer comprises a tail.

33. The kit of claim 32, wherein said tail is GC-rich.

34. The kit of claim 32, wherein said tail is between 2-30 nucleotides long.

35. The kit of claim 32, wherein said tail is at the 5′

end of said allele-specific primer.

36. The kit of claim 30, wherein said allele-specific primer has a melting temperature (Tm) between 50°

C. to 55°

C.

37. The kit of claim 30, wherein said allele-specific primer is at a concentration between 20-900 nM.

38. The kit of claim 30, wherein said allele-specific primer is designed to comprise a highly discriminating base at the 3′

terminus.

39. The kit of claim 30, wherein the 3′

-most nucleotide of said allele-specific primer is complementary to said first allelic variant.

40. The kit of claim 39, wherein said 3′

-most nucleotide is A if T is said first allelic variant;

or G if C is said first allelic variant.

41. The kit of claim 30, wherein said first allele-specific primer and/or first allele-specific blocker probe comprises at least one modified base.

42. The kit of claim 41, wherein said modified base is a 8-aza-7-deaza-dN (ppN) base analog, where N is adenine (A), cytosine (C), guanine (G), or thymine (T).

43. The kit of claim 41, wherein said modified base is a locked nucleic acid (LNA) base.

44. The kit of claim 41, wherein said modified base is any modified base that increases the Δ

Tm between matched and mismatched target sequences.

45. The kit of claim 30, wherein said allele-specific blocker probe further comprises an MGB moiety at the 5′

-end and/or at an internal position within said allele-specific blocker probe.

46. The kit of claim 45, wherein said allele-specific blocker probe comprises a 3′

end that is non-extendable.

47. The kit of claim 31, wherein said locus-specific detector probe further comprises a minor groove binder.

48. The kit of claim 30, wherein at least one of the two labels comprises a reporter moiety.

49. The kit of claim 31, further comprising a minor grove binder covalently attached to the quencher.

50. The kit of claim 30, wherein said kit further comprises a polymerase, dNTPs, and/or other reagents or buffers.

51. The kit of claim 50, wherein said polymerase is thermostable.

52. The kit of claim 30 or 31, wherein said kit is used to detect mutations in Ras, EGFR, Kit, pTEN, and/or p53.

53. The kit of claim 52, wherein said Ras mutation is a KRAS or an NRAS mutation.

54. The kit of claim 53, wherein said KRAS mutation is in codon 12 and/or codon 13 as depicted in FIG. 6.