Isothermal amplification of nucleic acids on a solid support
First Claim
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1. A method for amplifying nucleic acid molecules comprising:
- a) providing a plurality of immobilized first single stranded nucleic acid molecules and a plurality of immobilized primers, wherein the plurality of immobilized primers comprises first and second primers;
b) allowing the immobilized first single stranded nucleic acid molecules to anneal to first primers of the plurality of immobilized primers;
c) extending the annealed first primers using the first single stranded nucleic acid molecules as templates to generate double stranded nucleic acid molecules comprising immobilized first and second single stranded nucleic acid molecules;
d) denaturing the double stranded nucleic acid molecules to separate the immobilized first and second single stranded nucleic acid molecules;
e) annealing the first single stranded nucleic acid molecules to first primers of the plurality of immobilized primers and annealing the second single stranded nucleic acid molecules to second primers of the plurality of immobilized primers;
f) extending the annealed first and second primers using the first and second single stranded nucleic acid molecules as templates to generate double stranded nucleic acid molecules; and
g) repeating steps d-f to generate multiple copies of the nucleic acid molecules, wherein steps d-f are performed at the same temperature.
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Abstract
Methods for isothermal amplification of nucleic acids by the means of a solid support are disclosed. These methods are useful for applications needing high throughput, in particular nucleic acid sequencing.
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20 Claims
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1. A method for amplifying nucleic acid molecules comprising:
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a) providing a plurality of immobilized first single stranded nucleic acid molecules and a plurality of immobilized primers, wherein the plurality of immobilized primers comprises first and second primers; b) allowing the immobilized first single stranded nucleic acid molecules to anneal to first primers of the plurality of immobilized primers; c) extending the annealed first primers using the first single stranded nucleic acid molecules as templates to generate double stranded nucleic acid molecules comprising immobilized first and second single stranded nucleic acid molecules; d) denaturing the double stranded nucleic acid molecules to separate the immobilized first and second single stranded nucleic acid molecules; e) annealing the first single stranded nucleic acid molecules to first primers of the plurality of immobilized primers and annealing the second single stranded nucleic acid molecules to second primers of the plurality of immobilized primers; f) extending the annealed first and second primers using the first and second single stranded nucleic acid molecules as templates to generate double stranded nucleic acid molecules; and g) repeating steps d-f to generate multiple copies of the nucleic acid molecules, wherein steps d-f are performed at the same temperature. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)
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