Method of amplification of nucleic acids

• US 6,737,253 B1
• Filed: 05/20/2002
• Issued: 05/18/2004
• Est. Priority Date: 04/30/1999
• Status: Expired due to Fees

• Current Assignee: Takara Shuzo Company Limited (Ningbo Junsheng Investment Group Co., Ltd.)
• Original Assignee: Takara Shuzo Company Limited (Ningbo Junsheng Investment Group Co., Ltd.)
• Inventors: Tillett, Daniel
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US09/581,822
• Time in Patent Office: 729 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/810, 536/24.33, 536/24.3, 935/76, 935/77, 935/78
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2549/113 using nested probes

Abstract

The present invention relates generally to genetic analysis and, in particular, to genomics. More specifically, the invention relates to a method of amplification of a nucleic acid (or a nucleotide sequence of interest) which may be applied to sequencing in general and, in particular, to genome sequencing.

Claims

1. A method of amplifying a nucleotide sequence of interest wherein the nucleotide sequence comprises at least one region of known sequence, wherein the region of known sequence comprises, in a 3′

to 5′

direction;

a first known region and a second known region wherein the first and second known regions are immediately adjacent each other and wherein the method comprises;

1) a first amplification step of at least two cycles comprising amplifying the nucleotide sequence of interest using at least

(a) as a template, a sequence comprising at least the nucleotide sequence of interest;

(b) one first primer having, in a 5′

to 3′

direction;

a 5′

tag sequence;

a degenerate sequence corresponding to the first known region; and

a sequence complementary to the second known region of the nucleotide sequence of interest; and

(c) a reverse primer, deoxyribonucleoside triphosphates (dNTPs) and suitable enzyme conducted under reaction conditions such that the first amplification step generates a first amplification product; and

2) a second amplification step comprising amplifying the nucleotide sequence of interest using at least

(a) as a template, the first amplification product; and

(b) one second primer having, in a 5′

to 3′

direction;

a sequence which hybridises to the complementary strand of the 5′

tag sequence of the first primer and a sequence complementary to the first known region of the nucleotide sequence of interest;

which second amplification step generates a second amplification product.

2. A method of amplifying and sequencing a nucleotide sequence of interest comprising amplifying a nucleotide sequence of interest according to the method of claim 1 and sequencing the second amplification product.

3. A method according to claim 1 wherein the amplification step is a polymerase chain reaction.

4. A method according to claim 1 wherein, in the first and second primers respectively, the 5′

tag sequence and the sequence which hybridises to the complementary strand of the 5′

tag sequence of the first primer, are the same.

5. A method according to claim 1 wherein the 5′

tag sequence is a vector specific sequence.

6. A method according to claim 5 wherein the 5′

tag sequence is derived from M13 phage, pUC18, pBR322, pGEM, BLUESCRIPT, or pBELOBACII.

7. A method according to claim 1 wherein the 5′

tag sequence is 10 to 25 bases in length.

8. A method according to claim 7 wherein the 5′

tag sequence is 15 to 18 bases in length.

9. A method according to claim 8 wherein the 5′

tag sequence is 15 bases in length.

10. A method according to claim 1 wherein, in the first primer, the sequence complementary to the second known region is 4 to 8 bases in length.

11. A method according to claim 10 wherein, in the first primer, the sequence complementary to the second known region is 6 bases in length.

12. A method according to claim 1 wherein, in the first primer, the degenerate sequence together with the sequence complementary to the second known region of the nucleotide sequence of interest is 6 to 12 bases in length.

13. A method according to claim 12 wherein, in the first primer, the degenerate sequence together with the sequence complementary to the second known region of the nucleotide sequence of interest is 9 bases in length.

14. A method according to claim 1 wherein, in the second primer, the sequence complementary to the first known region of the nucleotide sequence of interest is 1 to 5 bases in length.

15. A method according to claim 14 wherein, in the second primer, the sequence complementary to the first known region of the nucleotide sequence of interest is 3 bases in length.

16. A method according to claim 2 wherein the 5′

tag sequence, or a sequence which hybridises to the complementary sequence of the 5′

tag sequence, is used as a sequencing primer.

17. A method according to claim 2 wherein the second primer is used as a sequencing primer.

18. A method according to claim 1 wherein the first and second amplifications comprise two first primers and two second primers respectively, wherein one of the first primers acts as a reverse primer in the first amplification step and one of the second primers acts as a reverse primer in the second amplification step.

19. A method according to claim 18 wherein the 5′

tag sequence on each of the first primers is different.

20. A method according to claim 1 wherein steps 1) and 2) are performed simultaneously.

21. A set of primers comprising:

(a) a first primer having, in a 5′

to 3′

direction;

a 5′

tag sequence;

a degenerate sequence; and

a predetermined sequence, wherein the sequences are immediately adjacent to each other; and

(b) a second primer having in a 5′

to 3′

direction;

a sequence which hybridises to the complementary strand of the 5′

tag sequence of the first primer, and a predetermined sequence corresponding to at least a portion of the degenerate sequence of the first primer.

22. A primer set according to claim 21 wherein, the predetermined sequence of the first primer is 4 to 8 bases in length.

23. A primer set according to claim 22 wherein, the predetermined sequence of the first primer is 6 bases in length.

24. A primer set according to claim 21 wherein, the degenerate sequence together with the predetermined sequence of the first primer is 6 to 12 bases in length.

25. A primer set according to claim 24 wherein, the degenerate sequence together with the predetermined sequence of the first primer is 9 bases in length.

26. A primer set according to claim 21 wherein, the predetermined sequence of the second primer is 1 to 5 bases in length.

27. A primer set according to claim 26 wherein, the predetermined sequence of the second primer is 3 bases in length.

28. A primer set according to claim 21 wherein the 5′

tag sequence is a vector specific sequence.

29. A primer set according to claim 28 wherein the 5′

tag sequence is derived from M13 phage, pUC18, pBR322, pGEM, BLUESCRIPT, or pBELOBACII.

30. A primer set according to claim 21 wherein the 5′

tag sequence is 10 to 25 bases in length.

31. A primer set according to claim 30 wherein the 5′

tag sequence is 15 to 18 bases in length.

32. A primer set according to claim 31 wherein the 5′

tag sequence is 15 bases in length.

33. A primer set library comprising a set of primers according to claim 21.

34. A kit comprising a primer set according to claim 21.

35. A kit comprising a primer library according to claim 33.

36. A method of amplifying and sequencing a nucleotide sequence of interest wherein the nucleotide sequence comprises at least one region of known sequence, wherein the region of known sequence comprises, in a 3′

to 5′

direction;

a first known region of 3 bases and a second known region of 6 bases wherein the first and second known regions are immediately adjacent each other and wherein the method comprises;

1) a first polymerase chain reaction (PCR) of at least two cycles comprising at least

(a) as a template, a sequence comprising at least the nucleotide sequence of interest;

(b) one first primer having, in a 5′

to 3′

direction;

a 5′

tag sequence;

a degenerate sequence corresponding to the first known region; and

a sequence complementary to the second known region of the nucleotide sequence of interest; and

(c) a reverse primer, deoxyribonucleoside triphosphates (dNTPs) and polymerase conducted under reaction conditions such that the first PCR generates a first PCR product;

2) a second PCR comprising at least

(a) as a template, the first PCR product; and

(b) one second primer having, in a 5′

to 3′

direction;

the same 5′

tag sequence as the first primer and a sequence complementary to the first known region of the nucleotide sequence of interest which second PCR generates a second PCR product; and

3) sequencing the second PCR product using the 5′

tag sequence as a sequencing primer.

40. A method according to claim 36 wherein the 5′

tag sequence is a vector specific sequence.

42. A method according to claim 36 wherein the 5′

tag sequence is 10 to 25 bases in length.

43. A method according to claim 42 wherein the 5′

tag sequence is 15 to 18 bases in length.

44. A method according to claim 43 wherein the 5′

tag sequence is 15 bases in length.

37. A method of amplifying and sequencing a nucleotide sequence of interest wherein the nucleotide sequence comprises at least one region of known sequence, wherein the region of known sequence comprises, in a 3′

to 5′

direction;

a first known region and a second known region wherein the first and second known regions are immediately adjacent each other and wherein the method comprises;

1) an amplification step of at least two cycles comprising amplifying the nucleotide sequence of interest using at least

(a) as a template, a sequence comprising at least the nucleotide sequence of interest;

(b) one primer having, in a 5′

to 3′

direction;

a 5′

tag sequence;

a degenerate sequence corresponding to the first known region; and

a sequence complementary to the second known region of the nucleotide sequence of interest; and

(c) a reverse primer, deoxyribonucleoside triphosphates (dNTPs) and suitable enzyme under reaction conditions such that the amplification step generates an amplification product; and

2) a sequencing step comprising sequencing the amplification product using;

(a) as a template, the amplification product; and

a sequencing primer having, in a 5′

to 3′

direction;

a sequence which hybridises to the complementary strand of the 5′

tag sequence of the primer utilised in the amplification step, and a sequence complementary to the first known region of the nucleotide sequence of interest.

38. A method according to claim 37 wherein the amplification step is a polymerase chain reaction.

39. A method according to claim 37 wherein, in the primer utilised in the amplification step and in the sequencing primers respectively, the 5′

tag sequence and the sequence which hybridises to the complementary strand of the 5′

tag sequence of the primer utilised in the amplification step, and the same.

41. A method according to claim 39 wherein the 5′

tag sequence is derived from M13 phage, pUC18, pBR322, pGEM, BLUESCRIPT, or pBELOBACII.

45. A method according to claim 37 wherein, in the primer utilised in the amplification step, the sequence complementary to the second known region is 4 to 8 bases in length.

46. A method according to claim 45 wherein, in the primer utilised in the amplification step, the sequence complementary to the second known region is 6 bases in length.

47. A method according to claim 37 wherein, in the primer utilised in the amplification step, the degenerate sequence together with the sequence complementary to the second known region of the nucleotide sequence of interest is 6 to 12 bases in length.

48. A method according to claim 47 wherein, in the primer utilised in the amplification step, the degenerate sequence together with the sequence complementary to the second known region of the nucleotide sequence of interest is 9 bases in length.

49. A method according to claim 38 wherein, in the sequencing primer, the sequence complementary to the first known region of the nucleotide sequence of interest is 1 to 5 bases in length.

50. A method according to claim 49 wherein, in the sequencing primer, the sequence complementary to the first known region of the nucleotide sequence of interest is 3 bases in length.