METHODS FOR SUPPRESSION PCR
First Claim
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.
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Accused Products
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.
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Citations
59 Claims
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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:
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(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. - View Dependent Claims (41, 42, 43, 44, 45, 46, 47, 48)
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2-40. -40. (canceled)
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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:
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(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. - View Dependent Claims (50, 51, 52, 53, 54, 55, 56)
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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. - View Dependent Claims (58, 59)
- at least one forward selective primer, the forward selective primer comprising;
Specification