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 generating two or more substantially monoclonal populations of template polynucleotides, comprising:
(a) contacting single-stranded template polynucleotides with a plurality of supports wherein each support comprises a plurality of first primers under annealing conditions optimized to generate supports having only one single-stranded template polynucleotide attached by hybridization thereto, and optionally extending the first primer in a template-dependent polymerization reaction to generate double-stranded template polynucleotides attached to the supports and optionally separating the strands of the double-stranded template polynucleotides attached to the supports;
(b) distributing the supports having a single-stranded template polynucleotide, or optionally a double-stranded template polynucleotide, attached thereto into separate individual reaction sites within an array of reaction sites; and
(c) forming two or more substantially monoclonal nucleic acid populations by amplifying the template polynucleotide at each reaction site, comprising:
i) providing a recombinase and a polymerase having a processivity of 100 base pairs or longer, and a second oligonucleotide primer in solution, wherein the second oligonucleotide primer in solution comprises an affinity moiety; and
ii) clonally amplifying 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
wherein the reaction sites are in continuous liquid phase communication with each other during the amplifying and wherein the continuous liquid phase further comprises a binding partner that interacts with the affinity moiety.
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 template polynucleotides attached to the supports comprise an affinity moiety.
13. The method of claim 1 wherein a binding partner moiety to which the affinity moiety binds, is attached to a paramagnetic bead, capable to form purification complexes.
14. The method of claim 13 further comprising attraction of the resulting amplification products with a magnet, thereby removing purification complexes.
15. The method of claim 1 , wherein the affinity moiety comprises a biotin moiety and the binding partner comprises an avidin-like moiety.
16. The method of claim 1 further comprising:
(a) loading at least two different template polynucleotides into separate individual reaction chambers in an array of reaction chambers;
(b) performing two different rounds of amplification within the reaction chambers comprising:
i. a first amplification reaction, wherein the reaction chambers are contacted with one or more reagents comprising a drag compound comprising a receptor moiety, and
ii. a second amplification, comprising contacting the reaction chambers with an amplification primer that hybridizes to at least a portion of one strand of the template polynucleotides and wherein the amplification primer comprises an affinity moiety that interacts with the receptor moiety; thereby amplifying the template polynucleotides within the reaction chambers and forming at least two substantially monoclonal nucleic acid populations, wherein the reaction chambers are in fluid communication with each other during amplifying.
17. The method of claim 16 wherein the affinity moiety comprises biotin and the receptor moiety comprises an avidin-like moiety.
18. The method of claim 16 wherein the second amplification does not include contacting reaction chambers with a drag compound.
19. The method of claim 16 wherein a binding partner moiety to which the affinity moiety binds, is attached to a paramagnetic bead, capable to form purification complexes.
20. The method of claim 19 further comprising attraction of the resulting amplification products with a magnet, thereby removing purification complexes.