Nucleic acid amplification
First Claim
1. A method for amplifying a plurality of nucleic acid templates, comprising:
- a) providing a plurality of forward primers immobilized on a support, wherein the plurality of forward primers includes a first forward primer and a second forward primer, and wherein the plurality of forward primers have substantially identical sequences;
b) providing a nucleic acid reverse strand from the plurality of nucleic acid templates, having a forward primer-binding sequence that can hybridize to any one of the plurality of forward primers;
c) hybridizing the first forward primer to the forward primer-binding sequence on the nucleic acid reverse strand;
d) generating an extended forward strand that is substantially complementary to the nucleic acid reverse strand and is hybridized thereto, by primer extension of the first forward primer using the reverse strand as a template, wherein the first forward primer becomes incorporated into the extended forward strand;
e) denaturing at least a portion of the extended forward strand comprising the incorporated first forward primer and the forward primer-binding sequence on the nucleic acid reverse strand and hybridizing the second forward primer to the forward primer-binding sequence on the nucleic acid reverse strand;
f) generating another extended forward strand that is substantially complementary to the reverse strand and is hybridized thereto, by primer extension of the second forward primer using the reverse strand as a template, wherein the second forward primer becomes incorporated into the extended forward strand; and
g) amplifying the plurality of nucleic acid templates simultaneously in a single continuous liquid phase without first compartmentalizing, by performing one or more amplification cycles comprising steps (e)-(f) under isothermal conditions to form clonal or substantially clonal nucleic acid populations, wherein the incorporated second forward primer of step (e) of an amplification cycle acts as the incorporated first forward primer of step (e) of a subsequent amplification cycle and the second forward primer of step (e) in the subsequent amplification cycle is a new second forward primer that has not undergone primer extension; and
wherein the amplifying is carried out using a recombinase and a polymerase.
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Accused Products
Abstract
In some embodiments, the present teachings provide methods for nucleic acid amplification, comprising forming a reaction mixture, and subjecting the reaction mixture to conditions suitable for nucleic acid amplification. In some embodiments, methods for nucleic acid amplification include subjecting the nucleic acid to be amplified to partially denaturing conditions. In some embodiments, methods for nucleic acid amplification include amplifying without fully denaturing the nucleic acid that is amplified. In some embodiments, the methods for nucleic acid amplification employ an enzyme that catalyzes homologous recombination and a polymerase. In some embodiments, methods for nucleic acid amplification can be conducted in a single reaction vessel. In some embodiments, methods for nucleic acid amplification can be conducted in a single continuous liquid phase of a reaction mixture, without need for compartmentalization of the reaction mixture or immobilization of reaction components. In some embodiments, methods for nucleic acid amplification comprise a amplifying at least one polynucleotide onto a surface under isothermal amplification conditions, optionally in the presence of a polymer. The polymer can include a sieving agent and/or a diffusion-reducing agent.
95 Citations
9 Claims
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1. A method for amplifying a plurality of nucleic acid templates, comprising:
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a) providing a plurality of forward primers immobilized on a support, wherein the plurality of forward primers includes a first forward primer and a second forward primer, and wherein the plurality of forward primers have substantially identical sequences; b) providing a nucleic acid reverse strand from the plurality of nucleic acid templates, having a forward primer-binding sequence that can hybridize to any one of the plurality of forward primers; c) hybridizing the first forward primer to the forward primer-binding sequence on the nucleic acid reverse strand; d) generating an extended forward strand that is substantially complementary to the nucleic acid reverse strand and is hybridized thereto, by primer extension of the first forward primer using the reverse strand as a template, wherein the first forward primer becomes incorporated into the extended forward strand; e) denaturing at least a portion of the extended forward strand comprising the incorporated first forward primer and the forward primer-binding sequence on the nucleic acid reverse strand and hybridizing the second forward primer to the forward primer-binding sequence on the nucleic acid reverse strand; f) generating another extended forward strand that is substantially complementary to the reverse strand and is hybridized thereto, by primer extension of the second forward primer using the reverse strand as a template, wherein the second forward primer becomes incorporated into the extended forward strand; and g) amplifying the plurality of nucleic acid templates simultaneously in a single continuous liquid phase without first compartmentalizing, by performing one or more amplification cycles comprising steps (e)-(f) under isothermal conditions to form clonal or substantially clonal nucleic acid populations, wherein the incorporated second forward primer of step (e) of an amplification cycle acts as the incorporated first forward primer of step (e) of a subsequent amplification cycle and the second forward primer of step (e) in the subsequent amplification cycle is a new second forward primer that has not undergone primer extension; and
wherein the amplifying is carried out using a recombinase and a polymerase. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9)
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Specification