Isothermal amplification of nucleic acids on a solid support
View Patent ↗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.
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.
2. The method of claim 1 , wherein the first single stranded nucleic acid molecules comprise common sequences at the 5′ and 3′ ends.
3. The method of claim 2 , wherein the first single stranded nucleic acid molecules comprise a common sequence Y at the 5′ end and a common sequence Z at the 3′ end.
4. The method of claim 3 , wherein the primers comprise sequence X, which is hybridizable to sequence Z.
5. The method of claim 4 , wherein the primer sequence X is the same as sequence Y.
6. The method of claim 1 , wherein the first primers comprise sequence X′ and the second primers comprise sequence X″.
7. The method of claim 6 , wherein sequence X′ is hybridizable to sequence Z.
8. The method of claim 7 , wherein the second single stranded nucleic acid molecules comprise a sequence at the 3′ end that is hybridizable to the second primers.
9. The method of claim 1 , wherein the nucleic acid molecules and primers are immobilized on a solid surface.
10. The method of claim 9 , wherein step (g) produces colonies of different nucleic acid molecules.
11. The method of claim 1 , wherein the colonies are generated at a density of 1000-100000 per mm 2 .
12. The method of claim 1 , wherein either or both of the first and second primers comprise a modification that facilitates release of at least a portion of the primer.
13. The method of claim 1 , further comprising releasing one or more of the immobilized first or second single stranded nucleic acid molecules.
14. The method of claim 1 , further comprising determining the sequence of one or more of the first or second nucleic acid molecules.
15. The method of claim 14 , wherein the sequence is determined by incorporation of labeled nucleotides.
16. The method of claim 15 , wherein the labeled nucleotides are incorporated onto the immobilized primers.
17. The method of claim 15 , wherein the labeled nucleotides are incorporated onto non-immobilized primers hybridized to the first or second single stranded nucleic acid molecules.
18. The method of claim 15 , wherein the label is a fluorescent group or a radioactive label.
19. The method of claim 14 , wherein the sequence is determined by incorporation of labeled oligonucleotides.
20. The method of claim 19 , wherein the label is a fluorescent group or a radioactive label.