US 9,518,292 B2Granted

Methods for suppression PCR

AssigneeBrandeis University

InventorsKen Sugino, Serena David, Saori Kato, Sean O'Toole, Sacha Nelson

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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.

Claims17

Dependent claims

A method of selectively amplifying 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 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 based on the selective amplification.

5 more elements

An assay for selectively amplifying 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 suppression target sequence, and (ii) a 5′ end suppression sequence that is at least 90% 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 at least 90% 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 based on the selective amplification.

5 more elements

Dependencies are inferred from the claim text.

Facts

Application: 14/119,471Filed 25 May 2012 · us provisional application
Priority: 26 May 2011
Published: US 2014/0315211 A123 Oct 2014
Granted: 13 Dec 2016
Term ends: Not estimatedThe source records don’t support an estimate.

Classification3 codes

C12Q 1/686
Nucleic acid amplification reactions: polymerase chain reaction [PCR]
C12Q 1/6858
Nucleic acid amplification reactions: allele-specific amplification
C12Q
Measuring or testing processes involving enzymes, nucleic acids or microorganisms

Family

Application61/490,341Us provisional application · filed 26 May 2011
US 2014/0315211 A1Pre-grant publication · published 23 Oct 2014
US 9,518,292 B2This patent

From the US filing history. Foreign family members aren’t listed.

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