Methods, compositions, systems, apparatuses and kits for nucleic acid amplification
Novel methods of generating a localized population of immobilized clonal amplicons on a support are provided.
1. A method of generating a clonal population of template nucleic acids, comprising:
(a) providing a support comprising multiple immobilized primers a first duplex template nucleic acid attached to the support, and non-immobilized primers, wherein:
the first duplex template nucleic acid comprises a first partner strand comprising an extended first immobilized primer generated by template-dependent extension of the first immobilized primer, and a second partner strand hybridized thereto wherein:
the second partner strand comprises a first primer-binding sequence complementary to the immobilized primers, and
the first partner strand comprises a second primer-binding sequence comprising nucleotides complementary to a non-immobilized primers, wherein the second primer-binding sequence is breathable;
(b) partially dissociating the first duplex template nucleic acid at the end containing the second primer-binding sequence and hybridizing a non-immobilized primer to the first partner strand;
(c) extending the hybridized non-immobilized primer to generate an extended non-immobilized primer strand while displacing the second partner strand;
(d) hybridizing the displaced second partner strand to a second immobilized primer on the support;
(e) extending the second immobilized primer on the support to generate a second duplex template nucleic acid comprising the second partner strand and an extended second immobilized primer strand; and
(f) repeating steps (b)-(e) for each duplex nucleic acid attached to the support to generate a clonal population of template nucleic acids attached to the support, wherein the method is performed under isothermal conditions;
wherein partial dissociating of duplex templates and hybridizing of non-immobilized primer comprises breathing of the second primer binding sequences.
2. The method of claim 1 , wherein partially dissociating the duplex nucleic acid is conducted in the presence of a recombinase.
3. The method of claim 1 , wherein the support is a solid support.
4. The method of claim 1 , wherein the support comprises beads or particles.
5. The method of claim 1 , wherein extension of a non-immobilized primer hybridized to a partially dissociated nucleic acid is conducted in the presence of a polymerase having strand-displacing activity.
6. The method of claim 1 , wherein the method is performed in a well.
7. The method of claim 1 , wherein the method is performed in an array.
8. The method of claim 1 , further comprising generating the first duplex template nucleic acid attached to the support of step (a) by contacting a template nucleic acid comprising a primer-binding sequence complementary to the immobilized primer with a support comprising the immobilized primers, annealing the template nucleic acid to the immobilized primer, and extending the immobilized primer by template-dependent extension.
9. The method of claim 8 , wherein a plurality of different template nucleic acids comprising a primer-binding sequence complementary to the immobilized primers are contacted with a plurality of supports comprising multiple immobilized primers in a single continuous liquid phase and wherein the ratio of the number of template nucleic acids to the total number of supports is selected to maximize the number of supports having only one template nucleic acid attached thereto, thereby generating a plurality of different first duplex template nucleic acids separately attached to a plurality of supports.
10. The method of claim 9 , wherein the plurality of different first duplex template nucleic acids separately attached to a plurality of supports are distributed individually into separate wells of an array of wells.
11. The method of claim 1 , further comprising generating the first duplex template nucleic acid attached to the support of step (a) by contacting a single-stranded nucleic acid that is an extended support-immobilized primer strand comprising a sequence of nucleotides complementary to a non-immobilized primer with a non-immobilized primer, annealing the extended support-immobilized primer strand to a non-immobilized primer, and extending the annealed non-immobilized primer by template-dependent extension.
12. The method of claim 11 , wherein the single-stranded nucleic acid that is an extended support-immobilized primer strand comprising a sequence of nucleotides complementary to a non-immobilized primer is generated by contacting a template nucleic acid comprising a primer-binding sequence complementary to the immobilized primer with a support comprising immobilized primers, annealing the template nucleic acid to the immobilized primer, extending the immobilized primer by template-dependent extension and separating the extended immobilized primer from the template nucleic acid.
13. The method of claim 11 , wherein a plurality of different single-stranded nucleic acids that are an extended support-immobilized primer strand comprising a sequence of nucleotides complementary to a non-immobilized primer are generated and are separately attached to a plurality of supports, and the plurality of supports are distributed individually into separate wells of an array of wells and wherein the plurality of supports are contacted with the non-immobilized primers in the wells.
14. The method of claim 1 , wherein a plurality of supports comprising multiple immobilized primers and a plurality of first duplex template nucleic acids that are separately attached to the plurality of supports are provided and wherein each first duplex template nucleic acid attached to a support is subjected to the method to generate a plurality of clonal populations of template nucleic acids attached to supports.
15. The method of claim 14 , wherein the method is performed for each first duplex template nucleic acid of the plurality of first duplex template nucleic acids that are separately attached to a plurality of supports in a single continuous liquid phase and the method is performed simultaneously for each first duplex template nucleic.
16. The method of claim 15 , wherein each first duplex template nucleic acid of the plurality of first duplex template nucleic acids is different.
17. The method of claim 1 , wherein all the immobilized primers are substantially identical in sequence or have an identical 3′ portion.
18. The method of claim 1 , further comprising subjecting template nucleic acids in the clonal population of template nucleic acids to a nucleic acid sequencing reaction.
19. The method of claim 14 , further comprising subjecting template nucleic acids in the clonal populations of template nucleic acids to a nucleic acid sequencing reaction.
20. The method of claim 19 , wherein template nucleic acids in each of a plurality of clonal populations of template nucleic acids are subjected to a nucleic acid sequencing reaction in parallel.