Claims |
What is claimed is: |
1. A method of amplifying a nucleotide s tide sequence comprises at least one reg region of known sequence comprises, in a region and a second known region wherein are immediately adjacent each other and |
equence of interest wherein the nucleo- ion of known sequence, wherein the 3' to 5' direction: a first known the first and second known regions wherein the method comprises: |
1) a first amplification step of at leas the nucleotide sequence of interest usin |
t two cycles comprising amplifying g at least |
(a) as a template, a sequence comprising of interest; |
at least the nucleotide sequence |
(b) one first primer having, in a 5' to degenerate sequence corresponding to the complementary to the second known region and |
3' direction: a 5' tag sequence; a first known region; and a sequence of the nucleotide sequence of interest; |
(c) a reverse primer, deoxyribonucleosid enzyme conducted under reaction conditio step generates a first amplification pro |
e triphosphates (dNTPs) and suitable ns such that the first amplification duct; and |
2) a second amplification step comprisin of interest using at least |
g amplifying the nucleotide sequence |
(a) as a template, the first amplificati |
on product; and |
(b) one second primer having, in a 5' to ises to the complementary strand of the and a sequence complementary to the firs nce of interest; which second amplificat ion product. |
3' direction: a sequence which hybrid- 5' tag sequence of the first primer t known region of the nucleotide seque- ion step generates a second amplificat- |
2. A method of amplifying and sequencing comprising amplifying a nucleotide seque of claim 1 and sequencing the second amp |
a nucleotide sequence of interest nce of interest according to the method lification product. |
3. A method according to claim 1 wherein chain reaction. |
the amplification step is a polymerase |
4. A method according to claim 1 wherein respectively, the 5' tag sequence and th complementary strand of the 5' tag seque 5. A method according to claim 1 wherein fic sequence. |
, in the first and second primers e sequence which hybridises to the nce of the first primer, are the same. the 5' tag sequence is a vector speci- |
6. A method according to claim 5 wherein M13 phage, pUC18, pBR322, pGEM, BLUESCRI |
the 5' tag sequence is derived from PT, or pBELOBACII. |
7. A method according to claim 1 wherein in length. |
the 5' tag sequence is 10 to 25 bases |
8. A method according to claim 7 wherein in length. |
the 5' tag sequence is 15 to 18 bases |
9. A method according to claim 8 wherein length. |
the 5' tag sequence is 15 bases in |
10. A method according to claim 1 wherei complementary to the second known region |
n, in the first primer, the sequence is 4 to 8 bases in length. |
11. A method according to claim 10 where complementary to the second known region |
in, in the first primer, the sequence is 6 bases in length. |
12. A method according to claim 1 wherei sequence together with the sequence comp of the nucleotide sequence of interest i |
n, in the first primer, the degenerate lementary to the second known region s 6 to 12 bases in length. |
13. A method according to claim 12 where sequence together with the sequence comp of the nucleotide sequence of interest i |
in, in the first primer, the degenerate lementary to the second known region s 9 bases in length. |
14. A method according to claim 1 wherei complementary to the first known region is 1 to 5 bases in length. |
n, in the second primer, the sequence of the nucleotide sequence of interest |
15. A method according to claim 14 where complementary to the first known region is 3 bases in length. |
in, in the second primer, the sequence of the nucleotide sequence of interest |
16. A method according to claim 2 wherei which hybridises to the complementary se used as a sequencing primer. |
n the 5' tag sequence, or a sequence quence of the 5' tag sequence, is |
17. A method according to claim 2 wherei ncing primer. |
n the second primer is used as a seque- |
18. A method according to claim 1 wherei comprise two first primers and two secon of the first primers acts as a reverse p and one of the second primers acts as a tion step. |
n the first and second amplifications d primers respectively, wherein one rimer in the first amplification step reverse primer in the second amplifica- |
19. A method according to claim 18 where the first primers is different. |
in the 5' tag sequence on each of |
20. A method according to claim 1 wherei aneously. |
n steps 1) and 2) are performed simult- |
21. A set of primers comprising: |
(a) a first primer having, in a 5' to 3' erate sequence; and a predetermined sequ ately adjacent to each other; and |
direction: a 5' tag sequence; a degen- ence, wherein the sequences are immedi- |
(b) a second primer having in a 5' to 3' to the complementary strand of the 5' ta a predetermined sequence corresponding t sequence of the first primer. |
direction: a sequence which hybridises g sequence of the first primer, and o at least a portion of the degenerate |
22. A primer set according to claim 21 w of the first primer is 4 to 8 bases in l |
herein, the predetermined sequence ength. |
23. A primer set according to claim 22 w of the first primer is 6 bases in length |
herein, the predetermined sequence . |
24. A primer set according to claim 21 w with the predetermined sequence of the f 25. A primer set according to claim 24 w with the predetermined sequence of the f 26. A primer set according to claim 21 w of the second primer is 1 to 5 bases in |
herein, the degenerate sequence together irst primer is 6 to 12 bases in length. herein, the degenerate sequence together irst primer is 9 bases in length. herein, the predetermined sequence length. |
27. A primer set according to claim 26 w of the second primer is 3 bases in lengt |
herein, the predetermined sequence h. |
28. A primer set according to claim 21 w specific sequence. |
herein the 5' tag sequence is a vector |
29. A primer set according to claim 28 w from M13 phage, pUC18, pBR322, pGEM, BLU |
herein the 5' tag sequence is derived ESCRIPT, or pBELOBACII. |
30. A primer set according to claim 21 w 25 bases in length. |
herein the 5' tag sequence is 10 to |
31. A primer set according to claim 30 w 18 bases in length. |
herein the 5' tag sequence is 15 to |
32. A primer set according to claim 31 w in length. |
herein the 5' tag sequence is 15 bases |
33. A primer set library comprising a se 34. A kit comprising a primer set accord |
t of primers according to claim 21. ing to claim 21. |
35. A kit comprising a primer library ac |
cording to claim 33. |
36. A method of amplifying and sequencin wherein the nucleotide sequence comprise wherein the region of known sequence com first known region of 3 bases and a seco the first and second known regions are i wherein the method comprises: |
g a nucleotide sequence of interest s at least one region of known sequence, prises, in a 3' to 5' direction: a nd known region of 6 bases wherein mmediately adjacent each other and |
1) a first polymerase chain reaction (PC at least |
R) of at least two cycles comprising |
(a) as a template, a sequence comprising of interest; |
at least the nucleotide sequence |
(b) one first primer having, in a 5' to degenerate sequence corresponding to the complementary to the second known region and |
3' direction: a 5' tag sequence; a first known region; and a sequence of the nucleotide sequence of interest; |
(c) a reverse primer, deoxyribonucleosid conducted under reaction conditions such PCR product; |
e triphosphates (dNTPs) and polymerase that the first PCR generates a first |
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 as the first primer and a sequence compl of the nucleotide sequence of interest w PCR product; and |
3' direction: the same 5' tag sequence ementary to the first known region hich second PCR generates a second |
3) sequencing the second PCR product usi primer. |
ng the 5' tag sequence as a sequencing |
37. A method of amplifying and sequencin wherein the nucleotide sequence comprise wherein the region of known sequence com first known region and a second known re known regions are immediately adjacent e ises: |
g a nucleotide sequence of interest s at least one region of known sequence, prises, in a 3' to 5' direction: a gion wherein the first and second ach other and wherein the method compr- |
1) an amplification step of at least two nucleotide sequence of interest using at |
cycles comprising amplifying the least |
(a) as a template, a sequence comprising of interest: |
at least the nucleotide sequence |
(b) one primer having, in a 5' to 3' dir sequence corresponding to the first know to the second known region of the nucleo |
ection: a 5' tag sequence; a degenerate n region; and a sequence complementary tide sequence of interest; and |
(c) a reverse primer, deoxyribonucleosid enzyme under reaction conditions such th an amplification product; and |
e triphosphates (dNTPs) and suitable at the amplification step generates |
2) a sequencing step comprising sequenci (a) as a template, the amplification pro in a 5' to 3' direction; a sequence whic strand of the 5' tag sequence of the pri step, and a sequence complementary to th sequence of interest. |
ng the amplification product using: duct; and a sequencing primer having, h hybridises to the complementary mer utilised in the amplification e first known region of the nucleotide |
38. A method according to claim 37 where ase chain reaction. |
in the amplification step is a polymer- |
39. A method according to claim 37 where amplification step and in the sequencing sequence and the sequence which hybridis the 5' tag sequence of the primer utilis the same. |
in, in the primer utilised in the primers respectively, the 5' tag es to the complementary strand of ed in the amplification step, and |
40. A method according to claim 36 where specific sequence. |
in the 5' tag sequence is a vector |
41. A method according to claim 39 where from M13 phage, pUC18, pBR322, pGEM, BLU |
in the 5' tag sequence is derived ESCRIPT, or pBELOBACII. |
42. A method according to claim 36 where bases in length. |
in the 5' tag sequence is 10 to 25 |
43. A method according to claim 42 where bases in length. |
in the 5' tag sequence is 15 to 18 |
44. A method according to claim 43 where in length. |
in the 5' tag sequence is 15 bases |
45. A method according to claim 37 where amplification step, the sequence complem is 4 to 8 bases in length. |
in, in the primer utilised in the entary to the second known region |
46. A method according to claim 45 where amplification step, the sequence complem is 6 bases in length. |
in, in the primer utilised in the entary to the second known region |
47. A method according to claim 37 where amplification step, the degenerate seque entary to the second known region of the 6 to 12 bases in length. |
in, in the primer utilised in the nce together with the sequence complem- nucleotide sequence of interest is |
48. A method according to claim 47 where amplification step, the degenerate seque entary to the second known region of the 9 bases in length. |
in, in the primer utilised in the nce together with the sequence complem- nucleotide sequence of interest is |
49. A method according to claim 38 where sequence complementary to the first know of interest is 1 to 5 bases in length. |
in, in the sequencing primer, the n region of the nucleotide sequence |
50. A method according to claim 49 where sequence complementary to the first know of interest is 3 bases in length. |
in, in the sequencing primer, the n region of the nucleotide sequence |