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 nucleic acid amplification, comprising:
(a) forming a reaction mixture by contacting, within a continuous liquid phase under nucleic acid synthesis conditions:
a plurality of different target polynucleotide templates in solution, each containing a first primer binding sequence and a second primer binding sequence, wherein the support contains only one type of primer;
a support containing multiple copies of a first universal primer that does not include any target-specific sequence and includes a sequence that is complementary or identical to the first primer-binding sequence, and the support does not include a primer containing a sequence that is complementary or identical to the first primer binding sequence, wherein the support contains only one type of primer;
multiple copies of a second universal primer in solution, wherein the second universal primer includes a sequence that complementary or identical to the second primer-binding sequence and is optionally pre-hybridized to one or more of the target polynucleotide templates; and
a polymerase; and
(b) forming two or more substantially monoclonal populations that are linked to the support, by clonally amplifying, by partially denaturing and without first compartmentalizing, within the same reaction mixture of step (a) in the continuous liquid phase, at least two of the target polynucleotide templates on the support using the first and second universal primers under isothermal amplification conditions.
2. The method of claim 1 , wherein the continuous liquid phase includes a dissolved cellulose polymer.
3. The method of claim 1 , wherein the reaction mixture includes at least 100 different target polynucleotide templates and wherein the method includes clonally amplifying at least 100 different target polynucleotide templates on different locations of the support.
4. The method of claim 1 , further including forming an array of monoclonal polynucleotide populations on the support.
5. The method of claim 1 , further including sequencing a polynucleotide of one or more of the substantially monoclonal populations.
6. The method of claim 5 , wherein the sequencing includes contacting the polynucleotide of the one or more of the substantially monoclonal populations with a sequencing reaction mixture including a polymerase.
7. The method of claim 6 , wherein the sequencing reaction mixture includes a sequencing primer having a sequence that is complementary or identical to a corresponding sequence within the first and/or second universal primers.
8. The method of claim 7 , further including incorporating a nucleotide into the sequencing primer.
9. The method of claim 8 , further including detecting a signal indicating the incorporating using a sensor.
10. The method of claim 9 , wherein the sensor includes a field effect transistor (FET).
11. The method of claim 9 , wherein the signal includes an increase in concentration of hydrogen, phosphate or pyrophosphate in the sequencing reaction mixture.
12. The method of claim 1 , wherein the clonally amplifying is performed without contacting the polynucleotide templates with a chemical denaturant during the clonally amplifying.
13. The method of claim 12 , wherein the chemical denaturant is selected from the group consisting of: urea, formamide, NaOH and guanidine-containing agents.
14. The method of claim 1 , wherein the reaction mixture includes a recombinase and wherein the clonally amplifying includes performing recombinase polymerase amplification.
15. The method of claim 14 , wherein the recombinase includes a wild-type, mutant, recombinant or variant uvsX protein.
16. The method of claim 14 , wherein the reaction mixture further includes a recombinase accessory factor.
17. The method of claim 16 , wherein the recombinase accessory factor includes a wild-type, mutant, recombinant or variant uvsY protein.