Nucleic acid amplification
The present disclosure provides methods, compositions, kits and systems for nucleic acid amplification. In some embodiments, nucleic acid amplification methods include subjecting the nucleic acid to be amplified to partially denaturing conditions. In some embodiments, nucleic acid amplification methods include amplifying without fully denaturing the nucleic acid that is amplified. In some embodiments, the nucleic acid amplification method employs 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 and/or 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 amplifying at least one polynucleotide onto a surface under isothermal amplification conditions, optionally in the presence of a polymer which can include a sieving agent and/or a diffusion-reducing agent.
1. A method for nucleic acid amplification comprising:
providing in a continuous liquid phase a plurality of supports each having a plurality of first oligonucleotide primers attached thereto, at least two nucleic acid templates, a recombinase and a polymerase having a processivity of 100 base pairs or longer, wherein the continuous liquid phase further comprises a second oligonucleotide primer in solution, wherein the second oligonucleotide primer in solution comprises an affinity moiety, wherein the continuous liquid phase further comprises a binding partner that interacts with the affinity moiety; and
clonally amplifying, within the continuous liquid phase, the at least two nucleic acid templates to form at least two substantially monoclonal populations of nucleic acids wherein at least 50% of the nucleic acids in each substantially monoclonal population share at least 80% sequence identity.
2. The method of claim 1 , wherein the polymerase is a T5 or T7 DNA polymerase having reduced exonuclease activity compared to wild-type T5 or T7 polymerase, and wherein if the polymerase is T7 polymerase, the reaction mixture further comprises thioredoxin.
3. The method of claim 2 , wherein the polymerase is a T7 DNA polymerase having a reduced 3′-5′ exonuclease activity, and wherein the T7 DNA polymerase has an E7A, a D5A, or both an E7A and a D5A mutation, wherein the numbering is relative to the amino acid sequence of SEQ ID NO: 1.
4. The method of claim 2 , wherein the polymerase is a T7 DNA polymerase selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4.
5. The method of claim 2 , wherein the continuous liquid phase further comprises a Bsu polymerase or a Sau polymerase.
6. The method of claim 1 , wherein the continuous liquid phase further comprises a recombinase accessory protein.
7. The method of claim 6 , wherein the recombinase is a UvsX protein and the recombinase accessory protein is a UvsY protein.
8. The method of claim 1 , wherein the continuous liquid phase comprises a single-stranded binding protein.
9. The method of claim 8 , wherein the single-stranded binding protein is gp32.
10. The method of claim 1 , wherein the continuous liquid phase further comprises a sieving agent.
11. The method of claim 1 , wherein the first oligonucleotide primers attached to the plurality of supports have an identical nucleotide sequence.
12. The method of claim 1 , wherein the interaction of the affinity moiety and the binding partner alters the mobility of a polynucleotide comprising the affinity through the continuous liquid phase.
13. The method of claim 1 , wherein the affinity moiety comprises a biotin moiety and the binding partner comprises an avidin-like moiety.
14. The method of claim 1 , wherein the plurality of supports are distributed on a plurality of sites on a surface.
15. The method of claim 14 , wherein the supports comprise beads or particles.
16. The method of claim 14 , wherein each site is independently operatively coupled to a sensor capable of detecting the presence of a nucleotide incorporation byproduct.
17. The method of claim 16 , wherein each sensor comprises a field effect transistor (FET).