METHODS FOR SUPPRESSION PCR

• US 20140315211A1
• Filed: 05/25/2012
• Published: 10/23/2014
• Est. Priority Date: 05/26/2011
• Status: Active Grant

• Current Assignee: Brandeis University
• Original Assignee: Brandeis University
• Inventors: Sugino, Ken, David, Serena, Kato, Saori, O'Toole, Sean, Nelson, Sacha
• Granted Patent: US 9,518,292 B2
• Time in Patent Office: Days
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2525/113 incorporating modified backbone
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2537/163 blocking probe

Abstract

Provided herein are approaches for the detection, identification, and/or selective amplification of specific target species or target variants of nucleic acid sequences, even within an excess of unwanted similar sequences or variants. These approaches include methods, assays, and kits for suppression PCR that require, in part, DNA polymerase that lacks 5′-3′ exonuclease activity, and a PCR primer, termed a forward selective primer or a nunchaku primer. The methods, assays, and kits provided herein are useful for a wide variety of applications, including cancer screening assays and kits, personalized screening assays, SNP (single nucleotide polymorphism) genotyping and identification, and downstream applications such as next generation high-throughput genomic sequencing and library construction.

Claims

1. A method of detecting the presence of one of two or more variants of a nucleic acid sequence in a nucleic acid sample, the method comprising:

(a) hybridizing a forward selective primer to a nucleic acid sample comprising an amplification target sequence and a suppression target sequence, wherein the forward selective primer comprises;

(i) a 3′

end priming sequence that is complementary to and hybridizes to a portion of the amplification target sequence and the at least one suppression target sequence, and (ii) a 5′

end suppression sequence that is substantially identical to a portion of the suppression target sequence, wherein the portion of the suppression target to which the 5′

suppression sequence is substantially identical is 5′

of the portion of the suppression target sequence to which the 3′

end priming sequence hybridizes;

(b) extending the hybridized forward selective primer of step (a) using a polymerase enzyme, the extension generating hybridized duplexes comprising;

(i) the amplification target sequence and a hybridized complementary extension sequence, wherein the complementary extension sequence comprises, in the 5′

to 3′

direction, the forward selective primer and a sequence complementary to the amplification target sequence, and (ii) the suppression target sequence and a hybridized complementary extension sequence, wherein the complementary extension sequence comprises, in the 5′

to 3′

direction, the forward selective primer and a sequence complementary to the suppression target sequence;

(c) denaturing the hybridized duplexes of step (b) to separate target sequences and complementary extension sequences; and

(d) hybridizing a reverse primer to the complementary extension sequences of step (c), and extending the hybridized reverse primer using a 5′

to 3′

polymerase that lacks 5′

to 3′

exonuclease activity and substantially lacks strand displacement activity, wherein if the complementary extension sequence comprises the sequence complementary to the suppression target sequence, then amplification is suppressed by formation of a stem loop by the 5′

suppression sequence and a shorter amplification product is generated, and wherein if the complementary extension sequence comprises the sequence complementary to the amplification target sequence, then amplification is not suppressed by formation of a stem loop and a longer amplification product is generated,

whereby the presence of one of two or more variants of a nucleic acid sequence is detected.

41. The method of claim 1, wherein steps (a)-(d) are repeated at least 10 times.

42. The method of claim 1, wherein the forward selective primer is at least 30 nucleotides in length.

43. The method of claim 1, wherein the longer amplification product generated in step (d) is at least 40 nucleotides in length.

44. The method of claim 1, wherein the extending of the hybridized reverse primer using the 5′

to 3′

polymerase that lacks 5′

to 3′

exonuclease activity and substantially lacks strand displacement activity of step (d) occurs for at least 5 seconds or more.

45. The method of claim 1, wherein the forward selective primer further comprises a loop spacer sequence, wherein said loop sequence is 5′

of the 3′

end priming sequence and does not comprise a sequence complementary to the complementary extension sequences.

46. The method of claim 1, wherein the reverse primer is a reverse selective primer.

47. The method of claim 1, wherein the forward selective primer comprises one or more locked nucleic acids (LNAs).

48. The method of claim 1, wherein the 5′

suppression sequence of the forward selective primer comprises one or more locked nucleic acids (LNAs).

2-40. -40. (canceled)

49. An assay for detecting the presence of one of two or more variants of a nucleic acid sequence in a nucleic acid sample, the assay comprising:

(a) hybridizing a forward selective primer to a nucleic acid sample comprising an amplification target sequence and a suppression target sequence, wherein the forward selective primer comprises;

(i) a 3′

end priming sequence that is complementary to and hybridizes to a portion of the amplification target sequence and the at least one suppression target sequence, and (ii) a 5′

end suppression sequence that is substantially identical to a portion of the suppression target sequence, wherein the portion of the at least one suppression target to which the 5′

suppression sequence is substantially identical is 5′

of the portion of the suppression target sequence to which the 3′

end priming sequence hybridizes;

(b) extending the hybridized forward selective primer of step (a) using a polymerase enzyme, the extension generating hybridized duplexes comprising;

(i) the amplification target sequence and a hybridized complementary extension sequence, wherein the complementary extension sequence comprises, in the 5′

to 3′

direction, the forward selective primer and a sequence complementary to the amplification target sequence, and (ii) the suppression target sequence and a hybridized complementary extension sequence, wherein the complementary extension sequence comprises, in the 5′

to 3′

direction, the forward selective primer and a sequence complementary to the suppression target sequence;

(c) denaturing the hybridized duplexes of step (b) to separate target sequences and complementary extension sequences; and

(d) hybridizing a reverse primer to the complementary extension sequences of step (c), and extending the hybridized reverse primer using a 5′

to 3′

polymerase that lacks 5′

to 3′

exonuclease activity and substantially lacks strand displacement activity, wherein if the complementary extension sequence comprises the sequence complementary to the suppression target sequence, then amplification is suppressed by formation of a stem loop by the 5′

suppression sequence and a shorter amplification product is generated, and wherein if the complementary extension sequence comprises the sequence complementary to the amplification target sequence, then amplification is not suppressed by formation of a stem loop and a longer amplification product is generated,

whereby the presence of one of two or more variants of a nucleic acid sequence is detected.

50. The assay of claim 49, wherein steps (a)-(d) are repeated at least 10 times.

51. The assay of claim 49, wherein the forward selective primer is at least 30 nucleotides in length.

52. The assay of claim 49, wherein the longer amplification product generated in step (d) is at least 40 nucleotides in length.

53. The assay of claim 49, wherein the extending of the hybridized reverse primer using the 5′

to 3′

polymerase that lacks 5′

to 3′

exonuclease activity and substantially lacks strand displacement activity of step (d) occurs for at least 5 seconds or more.

54. The assay of claim 49, wherein the forward selective primer further comprises a loop spacer sequence, wherein said loop spacer sequence is 5′

of the 3′

end priming sequence and does not comprise a sequence complementary to the complementary extension sequences.

55. The assay of assay of claim 49, wherein the assay is selected from a cancer screening assay, an autism screening assay, a prenatal genetic detection assay, and amicrobial detection assay.

56. The assay of assay of claim 49, wherein the amplification target sequence comprises a mutation or epigenetic modification found in cancer cells.

57. A kit for detecting the presence of one of two or more variants of a nucleic acid sequence in a nucleic acid sample, the kit comprising:

at least one forward selective primer, the forward selective primer comprising;

(i) a 3′

end priming sequence that is fully complementary and hybridizes to a portion of an amplification target sequence and a suppression target sequence, and (ii) a 5′

end suppression sequence that is substantially identical to a portion of the suppression target sequence, such that the portion of the suppression target to which the 5′

suppression sequence is substantially identical is 5′

of the portion of the suppression target sequence to which the 3′

end priming sequence hybridizes; and

instructions and packaging materials thereof.

58. The kit of claim 57, further comprising a reverse primer specific for a sequence complementary to both the amplification target sequence and the suppression target sequence.

59. The kit of claim 57, further comprising a reverse selective primer.