Method of nucleic acid amplification
A nucleic acid molecule can be annealed to an appropriate immobilized primer. The primer can then be extended and the molecule and the primer can be separated from one another. The extended primer can then be annealed to another immobilized primer and the other primer can be extended. Both extended primers can then be separated from one another and can be used to provide further extended primers. The process can be repeated to provide amplified, immobilized nucleic acid molecules. These can be used for many different purposes, including sequencing, screening, diagnosis, in situ nucleic acid synthesis, monitoring gene expression, nucleic acid fingerprinting, etc.
1. A method for amplifying nucleic acid molecules comprising:
a) providing a plurality of immobilized primers and immobilized first single stranded nucleic acid molecules;
b) allowing the immobilized first single stranded nucleic acid molecules to anneal to primers in the plurality of immobilized primers;
c) extending the primers that are annealed to the immobilized first single stranded nucleic acid molecules using the immobilized first single stranded nucleic acid molecules as template to provide second immobilized nucleic acid molecules;
d) separating the template and second immobilized nucleic acid molecules using a chemical denaturant;
e) allowing the template and second immobilized nucleic acid molecules to anneal to primers in the plurality of immobilized primers;
f) extending the primers that are annealed to the template and second immobilized nucleic acid molecules in suitable conditions; and
g) repeating steps d)-f) to generate multiple copies of the nucleic acid molecules.
2. The method of claim 1 , wherein the first single stranded nucleic acid molecules and primers are immobilized on a solid surface.
3. The method of claim 2 , wherein the primers are substantially homogenously dispersed over the solid surface.
4. The method of claim 2 , wherein the method produces a plurality of colonies on the solid support, wherein each colony represents a plurality of nucleic acid molecules comprising the same sequence.
5. The method of claim 1 , wherein, prior to step a), (i) non-immobilized single stranded nucleic acid molecules are annealed to primers in the plurality of immobilized primers; (ii) the annealed primers are extended using the annealed single stranded nucleic acid molecules as template to produce immobilized nucleic acid molecules; and (iii) the annealed single stranded nucleic acid molecules are separated from the immobilized nucleic acid molecules to produce the immobilized first single stranded nucleic acid molecules of step (a).
6. The method of claim 5 , wherein the non-immobilized single stranded nucleic acid molecules have different sequences.
7. The method of claim 1 , wherein the plurality of immobilized primers comprise at least two different sequences.
8. The method of claim 7 , wherein the immobilized single stranded nucleic acid molecules comprise a first, second and third part and wherein the first and second parts are located at the 5′ and 3′ ends of the immobilized single stranded nucleic acid molecules.
9. The method of claim 8 , wherein the third part is located between the first and second parts.
10. The method of claim 9 , wherein the third part is an unknown nucleic acid sequence.
11. The method of claim 10 , wherein the first part hybridizes to a primer of one sequence in the plurality of immobilized primers and the second part hybridizes to a primer of a different sequence in the plurality of immobilized primers.
12. The method of claim 7 , wherein in the plurality of immobilized primers the two different sequences are present in substantially the same concentrations.
13. The method of claim 1 , further comprising releasing one or more of the template or second immobilized nucleic acid molecules.
14. The method of claim 1 , further comprising determining the sequence of one or more of the nucleic acid molecules of step (g).
15. The method of claim 14 , wherein the second immobilized nucleic acid molecules are released and the sequence of the template is determined.
16. The method of claim 15 , wherein the sequencing is performed by extending primers hybridized to the template.
17. The method of claim 16 , wherein the sequencing is performed using labeled nucleotides.
18. The method of claim 17 , wherein the label is a fluorescent label.
19. The method of claim 1 , wherein the immobilized single stranded nucleic acid molecules of step (a) are different from one another.
20. The method of claim 19 , wherein the immobilized primers comprise the same sequence.
21. The method of claim 20 , wherein the method produces a plurality of different nucleic acid molecules.
22. The method of claim 21 , wherein the sequence of the plurality of different nucleic acid molecules is determined in parallel.
23. The method of claim 1 , wherein the chemical denaturant is an organic solvent.
24. The method of claim 23 , wherein the organic solvent is formamide.