Nucleic acid amplification
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.
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.
2. The method of claim 1 , wherein the support comprises only primers having sequences that are identical or substantially identical to the plurality of forward primers.
3. The method of claim 1 , further comprising generating an extended third forward strand that is a full-length complement of the reverse strand and is hybridized thereto, by primer extension of the third forward primer using the reverse strand as a template.
4. The method of claim 1 , further comprising:
as part of step e), hybridizing a reverse primer to a reverse primer-binding sequence on the extended forward strand, by denaturing the reverse primer-binding sequence on the extended forward strand from the nucleic acid reverse strand, wherein the reverse primer is a soluble primer; and
as part of step f) extending the reverse primer using the first forward strand as a template to generate an extended reverse strand that is a full-length complement of the forward strand and hybridized thereto.
5. The method of claim 1 , wherein the individual substantially clonal nucleic acid populations are immobilized within or on a different support from other substantially clonal nucleic acid populations.
6. The method of claim 1 , further comprising subjecting the extended forward strands to a sequencing reaction.
7. The method of claim 1 , wherein during step (e) a portion of the nucleic acid reverse strand remains hybridized to the extended first forward strand.
8. The method of claim 1 , wherein the extended forward strand and the nucleic acid reverse strand are not subjected to extreme denaturing conditions during step e).
9. The method of claim 8 , wherein the extended forward strand and the nucleic acid reverse strand are not contacted with chemical denaturants during step e).