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1. (WO2007010254) METHODS OF NUCLEIC ACID AMPLIFICATION AND SEQUENCING
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Claims

1. A method of amplifying one or more nucleic acid templates on a solid support which comprises:
a) bringing into contact the following components under conditions which permit a nucleic acid amplification reaction:
i) a solid support,
ii) a plurality of forward and reverse amplification primers, wherein the solid support is provided with the forward and/or reverse amplification primers immobilised thereon, and
iii) one or more nucleic acid templates to be amplified comprising at the 3 ' end a primer-binding sequence which is a sequence of nucleotides capable of annealing to the forward amplification primers and at the 5' end a primer-binding sequence which is a sequence of nucleotides the complement of which is capable of annealing to the reverse amplification primers,- and
b) carrying out a nucleic acid amplification reaction whereby said template (s) is/are amplified with said forward and reverse amplification primers,
characterised in that the amplification primers immobilised on the solid support comprise a template-specific portion which is a sequence of at least 26 consecutive nucleotides capable of annealing to a primer binding sequence in the template or the complement thereof and that the forward and reverse primers are not capable of annealing to any part of the template other than their respective primer binding sequences during the nucleic acid amplification reaction.

2. A method according to claim 1 wherein the one or more templates to be amplified each include a target sequence located between the two primer binding sequences, each said target sequence representing a fragment of the full sequence of a nucleic acid sample of interest, and the forward and reverse primers are selected based on knowledge of the full sequence of the nucleic acid sample of interest so as not to be capable of annealing to any part of the template other than their respective primer binding sequences during the nucleic acid amplification reaction. {"

3. A method according to claim 2 wherein the nucleic acid sample of interest is genomic DNA.

4. A method according to claim 3 wherein the nucleic acid sample of interest is human genomic DNA.

5. A method according to claim 4 wherein the nucleic acid sample of interest represents from 50% to 100% of the complete human genome.

6. A method according to any one of the preceding claims wherein the nucleic acid template (s) is/are produced by providing one or more target nucleic acid molecules to be amplified and adding thereto at the 3 ' end a first adaptor polynucleotide comprising a primer binding sequence capable of annealing to the forward amplification primers and at the 5 ' end a second adaptor polynucleotide comprising a primer binding sequence the complement of which is capable of annealing to the reverse amplification primers.

7. A method according to claim 6 wherein a plurality of templates to be amplified in a single amplification reaction are produced by providing a plurality of target nucleic acid molecules of different sequence and adding thereto at the 3 ' end a first universal adaptor polynucleotide comprising a sequence of nucleotides capable of annealing to the forward amplification primers and at the 5 ' end a second universal adaptor polynucleotide comprising a sequence of nucleotides the complement of which is capable of annealing to the reverse amplification primers.

8. A method according to claim 7 wherein the plurality of target nucleic acid molecules of different sequence are genomic DNA fragments .

9. A method according to claim 8 wherein the genomic DNA fragments are human genomic DNA fragments .

10. A method according to claim 9 wherein the sequences of the template-specific portions in the forward and reverse amplification primers are selected such that any sequence of 20 consecutive nucleotides in either template-specific portion is at least 2 bases different to any 20-mer in either strand of the human genome .

11. A method according to any preceding claim wherein both the forward and reverse amplification primers comprise a template-specific portion which is a sequence of at least 26 consecutive nucleotides .

12. A method according to any preceding claim wherein the template-specific portion in the forward and/or reverse amplification primers is a sequence of at least 30
consecutive nucleotides .

13. A method according to any preceding claim wherein the template-specific portion in the forward and/or reverse amplification primers is a sequence of at least 35
consecutive nucleotides .

14. A method according to any preceding claim wherein the template-specific portion in the forward and/or reverse amplification primers is a sequence of less than 50
consecutive nucleotides .

15. A method according to any one of claims 1 to 10 wherein the template-specific portion in the forward and/or reverse amplification primers is a sequence of from 30 to 45
consecutive nucleotides.

16. A method according to any one of claims 1 to 10 wherein the template-specific portion in the forward and/or reverse amplification primers is a sequence of from 35 to 40
consecutive nucleotides.

17. A method according to any one of claims 1 to 10 wherein the template-specific portion in the forward and/or reverse amplification primers is a sequence of 35 consecutive nucleotides

18. A method according to any one of the preceding claims wherein the forward and/or the reverse amplification primers immobilised on the solid support additionally comprise a linker portion which is not capable of annealing to the template to be amplified or the complement thereof.

19. A method according to claim 18 wherein the linker portion is a sequence of nucleotides which is not capable of annealing to the template to be amplified or the complement thereof .

20. A method according to claim 19 wherein the linker portion consists of from 1 to 20 consecutive nucleotides.

21. A method according to claim 20 wherein the linker portion consists of from 1 to 10 consecutive nucleotides

22. A method according to claim 20 or claim 21 wherein the linker portion consists of thymidine nucleotides.

23. A method according to claim 18 wherein the linker portion comprises a non-nucleotide chemical moiety.

24. A method according to any one of the preceding claims wherein in step a) the solid support is provided with both the forward and reverse amplification primers immobilised thereon.

25. A method according to claim 24 wherein in step a) the solid support is provided with both the forward and reverse amplification primers and the nucleic acid template to be amplified immobilised thereon, the template being attached to the solid support at the 5' end.

26. A method according to any one of the preceding claims wherein the forward and reverse primers are identical.

27. A method according to any one of the preceding claims wherein the solid support is a solid supported
polyacrylamide hydrogel .

28. Use of a method as defined in any one of claims 1 to 27 or the amplified products of said method for providing nucleic acid molecules for sequencing, re-sequencing, gene expression monitoring, genetic diversity profiling,
diagnosis, screening, whole genome sequencing, scoring or discovery of single nucleotide polymorphisms, or any other applications involving the amplification of nucleic acids or the sequencing thereof.

29. A method of nucleic acid sequencing which comprises amplifying one or more nucleic acid templates using a method as defined in any one of claims 1 to 27 and carrying out a sequencing reaction to determine the sequence of the whole or a part of at least one amplified nucleic acid strand produced in the amplification reaction.

30. A solid support having immobilised thereon a plurality of forward and/or reverse amplification primers,
characterised in that said forward and/or reverse
amplification primers comprise a template-specific portion capable of annealing to the template or the complement thereof which is a sequence of at least 26 consecutive nucleotides.

31. A solid support according to claim 30 wherein the template-specific portion in the forward and/or reverse amplification primers is a sequence of at least 30
consecutive nucleotides .

32. A solid support according to claim 31 wherein the template-specific portion in the forward and/or reverse amplification primers is a sequence of at least 35
consecutive nucleotides .

33. A solid support according to any one of claims 30 to 32 wherein the template-specific portion in the forward and/or reverse amplification primers is a sequence of less than 50 consecutive nucleotides.

34. A solid support according to claim 31 wherein the template-specific portion in the forward and/or reverse amplification primers is a sequence of from 30 to 45
consecutive nucleotides .

35. A solid support according to claim 32 wherein the template-specific portion in the forward and/or reverse amplification primers is a sequence of from 35 to 40
consecutive nucleotides .

36. A solid support according to claim 30 wherein the template-specific portion in the forward and/or reverse amplification primers is a sequence of 35 consecutive nucleotides

37. A solid support according to any one of claims 30 to 36 wherein the forward and/or the reverse amplification primers immobilised on the solid support additionally comprise a linker portion which is not capable of annealing to the template to be amplified or the complement thereof .

38. A solid support according to claim 37 wherein the linker portion is a sequence of nucleotides which is not capable of annealing to the template to be amplified or the complement thereof .

39. A solid support according to claim 38 wherein the linker portion consists of from 1 to 20 consecutive
nucleotides .

40. A solid support according to claim 39 wherein the linker portion consists of from 1 to 10 consecutive
nucleotides

41. A solid support according to claim 39 or claim 40 wherein the linker portion consists of thymidine
nucleotides.

42. A solid support according to claim 37 wherein the linker portion comprises a non-nucleotide chemical moiety.

43. A solid support according to any one of claims 30 to 32 wherein the forward and reverse primers are identical.

44. Use of the solid support of any one of claims 30 to 43 in a method of nucleic acid amplification.