US 9,175,337 B2Granted

Methods for amplifying nucleic acid using tag-mediated displacement

AssigneeGen-Probe Incorporated

InventorsNorman C. Nelson, Margarita Batranina-Kaminsky

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Abstract

Disclosed are methods for amplifying a nucleic acid target region using an amplification oligomer comprising a target-binding segment and a heterologous displacer tag situated 5′ to the target-binding segment. Initiation of an amplification reaction from the tagged amplification oligomer produces an amplicon comprising the displacer tag, such that once the complement of the displacer tag has been incorporated into a second amplicon, a displacer oligonucleotide having a sequence substantially corresponding to the displacer tag sequence is used to participate in subsequent rounds of amplification for displacement of an extension product primed from a site within the second amplicon 5′ to the displacer priming site. Also disclosed are related kits and reaction mixtures comprising the displacer-tagged amplification oligomer and corresponding displacer oligonucleotide.

Claims20

Dependent claims

A method of amplifying a nucleic acid target region, the method comprising:

contacting a target nucleic acid comprising the target region with
(1) a first amplification oligomer comprising
(a) a target-binding priming segment (T1) complementary to a 3′-end of the target region;
and
(b) a first heterologous displacer tag (D1) located 5′ to T1;
said contacting comprising conditions whereby the target nucleic acid serves as a template for extension from the first amplification oligomer to produce a first amplification product comprising T1 and D1;
(2) a second amplification oligomer comprising a target-binding segment T2 complementary to a region of the first amplicon that is the complement of a 5′-end of the target region, and wherein said contacting further comprises conditions whereby the first amplicon serves as a template to produce a second amplicon comprising segments cT1 and cD1, complementary to T1 and D1, respectively;
(3) a third amplification oligomer comprising target-binding priming segment T1p having a nucleotide sequence corresponding to T1, or corresponding to the complement of a second amplicon target sequence cT1′ near or overlapping with cT1 and situated 5′ to cD1;
and
(4) a fourth amplification oligomer comprising a displacer priming segment D1p having a nucleotide sequence corresponding to D1;
wherein said contacting further comprises conditions whereby the second amplicon serves as a template for extension from both the third and fourth amplification oligomers, wherein extension of T1p from a T1p:cT1/cT1′ hybrid produces a third amplicon, and wherein extension of D1p from a D1p:cD1 hybrid produces a fourth amplicon while displacing the third amplicon.

8 more elements

A method of amplifying a nucleic acid target region, the method comprising:

contacting a target nucleic acid comprising the target region with
(1) a first amplification oligomer comprising a target-binding priming segment (T1) complementary to a 3′-end of the target region;
said contacting comprising conditions whereby the target nucleic acid serves as a template for extension from the first amplification oligomer to produce a first amplicon;
(2) a second amplification oligomer comprising
(a) a target-binding segment T2 complementary to a region of the first amplicon that is the complement of a 5′-end of the target region;
and
(b) a first heterologous displacer tag (D1) located 5′ to T2;
said contacting further comprising conditions whereby the first amplicon serves as a template for amplification from the second amplification oligomer to produce a second amplicon comprising T2 and D1;
and whereby the second amplicon serves as a template for extension from the first amplification oligomer to produce a third amplicon comprising segments cT2 and cD1, complementary to T2 and D1, respectively;
(3) a third amplification oligomer comprising target-binding priming segment T2p having a nucleotide sequence corresponding to T2, or corresponding to the complement of a third amplicon target sequence cT2′ near or overlapping with cT2 and situated 5′ to cD1;
and
(4) a fourth amplification oligomer comprising a displacer priming segment D1p having a nucleotide sequence corresponding to D1;
wherein said contacting further comprises conditions whereby the third amplicon serves as a template for extension from both the third and fourth amplification oligomers, wherein extension of T2p from a T2p:cT2/cT2′ hybrid produces a fourth amplicon, and wherein extension of D1p from a D1p:cD1 hybrid produces a fifth amplicon while displacing the fourth amplicon.

10 more elements

A method of amplifying a nucleic acid target region, the method comprising:

(A) contacting a target nucleic acid comprising the target region with
(1) a first amplification oligomer comprising
(a) a target-binding priming segment (T1) complementary to a 3′-end of the target region;
(b) a heterologous universal tag (U1) located 5′ to T1;
and
(c) a first heterologous displacer tag (D1) located 5′ to U1;
said contacting comprising conditions whereby the target nucleic acid serves as a template for extension from the first amplification oligomer to produce a first amplicon comprising U1 and D1;
(2) a second amplification oligomer comprising
(a) a target-binding segment T2 complementary to a region of the first amplicon that is the complement of a 5′-end of the target region;
and
(b) optionally, a heterologous universal tag (U2) located 5′ to T2;
wherein said contacting further comprises conditions whereby the first amplicon serves as a template for amplification from the second amplification oligomer to produce a second amplicon comprising segments cU1 and cD1, complementary to U1 and D1, respectively, and optionally comprising U2;
(3) a third amplification oligomer comprising a universal priming segment U1p having a nucleotide sequence corresponding to U1;
(4) a fourth amplification oligomer comprising a displacer priming segment D1p having a nucleotide sequence corresponding to D1;
and
(5) if the second amplification oligomer comprises U2, a fifth amplification oligomer comprising a second universal priming segment U2p having a nucleotide sequence corresponding to U2;
wherein said contacting further comprises conditions whereby the second amplicon serves as a template for extension from both the third and fourth amplification oligomers, wherein extension of U1p from a U1p:cCU1 hybrid produces a third amplicon, and wherein extension of D1p from a D1p:cD1 hybrid produces a fourth amplicon while displacing the third amplicon;
or
(B) contacting a target nucleic acid comprising the target region with
(1) a first amplification oligomer comprising
(a) a target-binding priming segment (T1) complementary to a 3′-end of the target region;
and
(b) optionally, a heterologous universal tag (U2) located 5′ to T1;
said contacting comprising conditions whereby the target nucleic acid serves as a template for extension from the first amplification oligomer to produce a first amplicon;
(2) a second amplification oligomer comprising
(a) a target-binding segment T2 complementary to a region of the first amplicon that is the complement of a 5′-end of the target region;
(b) a heterologous universal tag (U1) located 5′ to T2;
and
(c) a first heterologous displacer tag (D1) located 5′ to U1;
said contacting further comprising conditions whereby the first amplicon serves as a template for amplification from the second amplification oligomer to produce a second amplicon comprising U1 and D1;
and whereby the second amplicon serves as a template for extension from the first amplification oligomer to produce a third amplicon comprising segments cU1 and cD1, complementary to U1 and D1, respectively;
(3) a third amplification oligomer comprising a universal priming segment U1p having a nucleotide sequence corresponding to U1;
(4) a fourth amplification oligomer comprising a displacer priming segment D1p having a nucleotide sequence corresponding to D1;
and
(5) if the first amplification oligomer comprises U2, a fifth amplification oligomer comprising a second universal priming segment U2p having a nucleotide sequence corresponding to U2;
wherein said contacting further comprises conditions whereby the third amplicon serves as a template for extension from both the third and fourth amplification oligomers, wherein extension of U1p from a U1p:cU1 hybrid produces a fourth amplicon, and wherein extension of D1p from a D1p:cD1 hybrid produces a fifth amplicon while displacing the fourth amplicon.

33 more elements

Dependencies are inferred from the claim text.

Facts

Application: 14/344,372Filed 21 Sep 2012 · us provisional application
Priority: 21 Sep 2011
Published: US 2014/0370550 A118 Dec 2014
Granted: 3 Nov 2015
Term ends: Not estimatedThe source records don’t support an estimate.

Classification6 codes

C12P 19/34
Nucleotides: polynucleotides, e.g. nucleic acids, oligoribonucleotides
C12Q 1/6855
Using modified primers or templates: ligating adaptors
C12Q 1/6844
Involving nucleic acids: nucleic acid amplification reactions
C12Q 2537/137
The purpose or use of: a displacement step