Annealing control primer and its uses

• US 20030175749A1
• Filed: 10/11/2002
• Published: 09/18/2003
• Est. Priority Date: 12/08/2001
• Status: Abandoned Application

• Current Assignee: Seegene, Inc.
• Original Assignee: Seegene, Inc.
• Inventors: Chun, Jong-Yoon
• Application Number:
  US10/269,031
• Publication Number:
  US 20030175749A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• 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 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine

Abstract

The present invention relates to an annealing control primer for improving annealing specificity in nucleic acid amplification and its applications to all fields of nucleic acid amplification-involved technology. The present primer comprises (a) a 3′-end portion having a hybridizing nucleotide sequence substantially complementary to a site on a template nucleic acid to hybridize therewith; (b) a 5′-end portion having a pre-selected arbitrary nucleotide sequence; and (c) a regulator portion positioned between said 3′-end portion and said 5′-end portion comprising at least one universal base or non-discriminatory base analog, whereby said regulator portion is capable of regulating an annealing portion of said primer in association with annealing temperature.

Claims

1. An annealing control primer for improving annealing specificity in nucleic acid amplification, which comprises:

(a) a 3′

-end portion having a hybridizing nucleotide sequence substantially complementary to a site on a template nucleic acid to hybridize therewith;

(b) a 5′

-end portion having a pre-selected arbitrary nucleotide sequence; and

(c) a regulator portion positioned between said 3′

-end portion and said 5′

-end portion comprising at least one universal base or non-discriminatory base analog, whereby said regulator portion is capable of regulating an annealing portion of said primer in association with annealing temperature.

2. The annealing control primer according to claim 1, wherein said pre-selected arbitrary nucleotide sequence of said 5′

-end portion is substantially not complementary to any site on said template nucleic acid.

3. The annealing control primer according to claim 1, wherein said template nucleic acid is gDNA, cDNA or mRNA.

4. The annealing control primer of claim 3, wherein said gDNA or cDNA is single or double-stranded DNA.

5. The annealing control primer according to claim 1, wherein said nucleic acid amplification is performed under a first and a second annealing temperatures.

6. The annealing control primer according to claim 5, wherein said first annealing temperature in said nucleic acid amplification is identical to or lower than said second annealing temperature.

7. The annealing control primer according to claim 5, wherein said 3′

-end portion is involved in annealing at said first annealing temperature and said 5′

-end portion serves as a priming site at said second annealing temperature.

8. The annealing control primer according to claim 5, wherein said regulator portion is capable of restricting said annealing portion of said primer to said 3′

-end portion at said first annealing temperature.

9. The annealing control primer according to claim 5, wherein said first annealing temperature is between about 30°

C. and 68°

C.

10. The annealing control primer according to claim 5, wherein said second annealing temperature is between about 50°

C. and 72°

C.

11. The annealing control primer according to claim 1, wherein said annealing control primer has a general formula of 5′

-X_p-Y_q-Z_r-3′

, wherein X_p represents said 5′

-end portion having said pre-selected arbitrary nucleotide sequence substantially not complementary to any site on the template nucleic acid;

Y_q represents said regulator portion comprising at least one universal base or non-discriminatory base analog;

Z_r represents said 3′

-end portion having a hybridizing nucleotide sequence substantially complementary to a site on the template nucleic acid to hybridize therewith;

wherein p, q and r represent the number of nucleotides; and

wherein X, Y and Z is deoxyribonucleotide or ribonucleotide.

12. The annealing control primer according to claim 11, wherein said universal base or non-discriminatory base analog is capable of forming base-pairs with each of the natural DNA/RNA bases with little discrimination between said natural DNA/RNA bases.

13. The annealing control primer according to claim 11, wherein said universal base or non-discriminatory base analog is selected from the group consisting of deoxyinosine, inosine, 7-deaza-2′

-deoxyinosine, 2-aza-2′

-deoxyinosine, 2′

-OMe inosine, 2′

-F inosine, deoxy 3-nitropyrrole, 3-nitropyrrole, 2′

-OMe 3-nitropyrrole, 2′

-F 3-nitropyrrole, 1-(2′

-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole, deoxy 5-nitroindole, 5-nitroindole, 2′

-OMe 5-nitroindole, 2′

-F 5-nitroindole, deoxy 4-nitrobenzimidazole, 4-nitrobenzimidazole, deoxy 4-aminobenzimidazole, 4-aminobenzimidazole, deoxy nebularine, 2′

-F nebularine, 2′

-F 4-nitrobenzimidazole, PNA-5-introindole, PNA-nebularine, PNA-inosine, PNA-4-nitrobenzimidazole, PNA-3-nitropyrrole, morpholino-5-nitroindole, morpholino-nebularine, morpholino-inosine, morpholino-4-nitrobenzimidazole, morpholino-3-nitropyrrole, phosphoramidate-5-nitroindole, phosphoramidate-nebularine, phosphoramidate-inosine, phosphoramidate-4-nitrobenzimidazole, phosphoramidate-3-nitropyrrole, 2′

-0-methoxyethyl inosine, 2′

0-methoxyethyl nebularine, 2′

-0-methoxyethyl 5-nitroindole, 2′

-O-methoxyethyl 4-nitro-benzimidazole, 2′

-0-methoxyethyl 3-nitropyrrole, and combinations thereof.

14. The annealing control primer according to claim 11, wherein said universal base or non-discriminatory base analog is deoxyinosine, 1-(2′

-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole or 5-nitroindole.

15. The annealing control primer according to claim 11, wherein said regulator portion comprises contiguous nucleotides having universal base or non-discriminatory base analog.

16. The annealing control primer according to claim 11, wherein said deoxyribonucleotide is naturally occurring dNMP, modified nucleotide and non-natural nucleotide.

17. The annealing control primer according to claim 11, wherein p represents an integer of 15 to 60.

18. The annealing control primer according to claim 11, wherein q is at least 2.

19. The annealing control primer according to claim 11, wherein q is at least 3.

20. The annealing control primer according to claim 11, wherein q represents an integer of 2 to 15.

21. The annealing control primer according to claim 11, wherein r represents an integer of 6 to 50.

22. The annealing control primer according to claim 11, wherein p is an integer of 15 to 60, q is an integer of 2 to 15 and r is an integer of 6 to 30.

23. The annealing control primer according to claim 11, wherein X_p comprises a universal primer sequence.

24. The annealing control primer according to claim 11, wherein X_p includes a sequence or sequences recognized by a restriction endonuclease or restriction endonucleases.

25. The annealing control primer according to claim 11, wherein X_p comprises at least one nucleotide with a label for detection or isolation.

26. The annealing control primer according to claim 11, wherein Z_r is a nucleotide sequence which hybridizes to the polyadenosine (polyA) tail of an mRNA.

27. The annealing control primer according to claim 26, wherein Z_r comprises at least 10 contiguous deoxythymidine nucleotides.

28. The annealing control primer according to claim 26, wherein Z_r comprises at least 10 contiguous deoxythymidine nucleotides having 3′

-V at its 3′

-end;

in which V is one selected from the group consisting of deoxyadenosine, deoxycytidine and deoxyguanosine.

29. The annealing control primer according to claim 26, wherein Z_r comprises at least 10 contiguous deoxythymidine nucleotides having 3′

-NV at its 3′

-end;

in which V is one selected from the group consisting of deoxyadenosine, deoxycytidine and deoxyguanosine, and N is one selected from the group consisting of deoxyadenosine, deoxythymidine, deoxycytidine and deoxyguanosine.

30. The annealing control primer according to claim 11, wherein Z_r is a nucleotide sequence substantially complementary to a target sequence in the template nucleic acid.

31. The annealing control primer according to claim 11, wherein Z_r is a random nucleotide sequence.

32. The annealing control primer according to claim 11, wherein Z_r is a nucleotide sequence substantially complementary to a consensus sequence found in a gene family.

33. The annealing control primer according to claim 11, wherein Z_r is a degenerate nucleotide sequence selected from a plurality of combinations of nucleotides encoding a predetermined amino acid sequence.

34. The annealing control primer according to claim 11, wherein Z_r comprises at least one ribonucleotide.

35. The annealing control primer according to claim 11, wherein Z_r comprises at least one nucleotide complementary to allelic site.

36. The annealing control primer according to claim 11, wherein Z_r comprises at least one mismatch nucleotide to a target nucleic acid for mutagenesis.

37. A kit comprising the primer or the primer set according to any one of claims 1-36.

38. The kit according to claim 37, wherein the kit further comprises a primer or a primer pair having a nucleotide sequence corresponding to the 5′

-end portion of said primer.

39. A method for amplifying a nucleic acid sequence from a DNA or a mixture of nucleic acids, wherein said method comprises performing an amplification reaction using primers, characterized in that at least one primer has the same structure as the primer of claim 1.

40. The method according to claim 39, wherein said method is performed using two stage amplifications, which comprises:

(a) performing a first-stage amplification of said nucleic acid sequence at a first annealing temperature comprising at least two cycles of primer annealing, primer extending and denaturing, using the primer pair of claim leach having at its 3′

-end portion a hybridizing sequence substantially complementary to a region of said nucleic acid sequence to hybridize therewith, under conditions in which each primer anneals to said region of said nucleic acid sequence, whereby the amplification product of said nucleic acid sequence is generated; and

(b) performing a second-stage amplification of said amplification product generated from step (a) at a second annealing temperature, which is high stringent conditions, comprising at least one cycle of primer annealing, primer extending and denaturing, using the same primers as used in step (a) or a primer pair each comprising a pre-selected arbitrary nucleotide sequence corresponding to each 5′

-end portion of said primers used in step (a), under conditions in which each primer anneals to the 3′

- and 5′

-ends of said amplification product, respectively, whereby said amplification product is re-amplified.

41. A method for selectively amplifying a target nucleic acid sequence from a DNA or a mixture of nucleic acids, wherein said method comprises performing an amplification reaction using primers, characterized in that at least one primer has the same structure as the primer of claim 1.

42. The method according to claim 41, wherein said method is performed using two stage amplifications, which comprises:

(a) performing a first-stage amplification of said target nucleic acid sequence at a first annealing temperature comprising at least two cycles of primer annealing, primer extending and denaturing, using the primer pair of claim 1 each having at its 3′

end portion a hybridizing sequence substantially complementary to a region of said target nucleic acid sequence to hybridize therewith, under conditions in which each primer anneals to its target nucleotide sequence, whereby the amplification product of said target nucleotide sequence is generated; and

(b) performing a second-stage amplification of said amplification product generated from step (a) at a second annealing temperature, which is high stringent conditions, comprising at least one cycle of primer annealing, primer extending and denaturing, using the same primers as used in step (a) or a primer pair each comprising a pre-selected arbitrary nucleotide sequence corresponding to each 5′

-end portion of said primers used in step (a), under conditions in which each primer anneals to the 3′

- and 5′

-ends of said amplification product, respectively, whereby said amplification product is re-amplified.

43. A method for selectively amplifying a target nucleic acid sequence from an mRNA, wherein said method comprises reverse transcribing said mRNA and performing an amplification reaction using primers, characterized in that at least one primer has the same structure as the primer of claim 1.

44. The method according to claim 43, wherein said method is performed using two stage amplifications, which comprises:

(a) contacting said mRNA with an oligonucleotide dT primer which is hybridized to polyA tail of said mRNA under conditions sufficient for template driven enzymatic deoxyribonucleic acid synthesis to occur;

(b) reverse transcribing said mRNA to which said oligonucloetide dT pirmer hybridizes to produce a first DNA strand that is complementary to said mRNA to which said oligonucloetide dT pirmer hybridizes;

(c) performing a first-stage amplification of said target nucleic acid sequence from said first DNA strand obtained from step (b) at a first annealing temperature comprising at least two cycles of primer annealing, primer extending and denaturing, using the primer pair of clai 1 each having at its 3′

end portion a hybridizing sequence substantially complementary to a region of said target nucleic acid sequence to hybridize therewith, under conditions in which each primer anneals to its target nucleotide sequence, whereby the amplification product of said target nucleotide sequence is generated; and

(d) performing a second-stage amplification of said amplification product generated from step (c) at a second annealing temperature, which is high stringent conditions, comprising at least one cycle of primer annealing, primer extending and denaturing, using the same primers as used in step (c) or a primer pair each comprising a pre-selected arbitrary nucleotide sequence corresponding to each 5′

-end portion of said primers used in step (c), under conditions in which each primer anneals to the 3′

- and 5′

-ends of said amplification product, respectively, whereby said amplification product is re-amplified.

45. A method for detecting DNA complementary to differentially expressed mRNA in two or more nucleic acid samples, wherein said method comprises reverse transcribing said mRNA and performing an amplification reaction using primers, characterized in that at least one primer has the same structure as the primer of claim 1.

46. The method according to claim 45, wherein said method is performed using two stage amplifications, which comprises:

(a) providing a first sample of nucleic acids representing a first population of mRNA transcripts and a second sample of nucleic acids representing a second population of mRNA transcripts;

(b) separately contacting each of said first nucleic acid sample and said second nucleic acid sample with a first primer of claim 1, in which the 3′

-end portion of said first primer comprises a hybridizing nucleotide sequence substantially complementary to a first site in said differentially expressed mRNA to hybridize therewith, under conditions sufficient for template driven enzymatic deoxyribonucleic acid synthesis to occur;

(c) reverse transcribing said differentially expressed mRNA to which said first primer hybridizes to produce a first population of first cDNA strands that are complementary to said differentially expressed mRNA in said first nucleic acid sample to which said first primer hybridizes, and a second population of first cDNA strands that are complementary to said differentially expressed mRNA in said second nucleic acid sample to which said first primer hybridizes;

(d) purifying and quantifying each of said first and second populations of first cDNA strands;

(e) performing a first-stage amplification of each of said first and second population of first DNA strands obtained from step (d) at a first annealing temperature comprising at least one cycle of primer annealing, primer extending and denaturing, using a second primer of claim 1 having at its 3 end portion a hybridizing sequence substantially complementary to a second site in said first and second populations of first cDNA strands, under conditions in which said second primer anneals to said second site in each population of said first cDNA strands, whereby first and second populations of second cDNA strands are generated;

(f) performing a second-stage amplification of each second cDNA strand generated from step (e) at a second annealing temperature, which is high stringent conditions, comprising at least two cycles of primer annealing, primer extending and denaturing, using the same first and second primers as used in steps (b) and (e), respectively, or a primer pair each comprising a pre-selected arbitrary nucleotide sequence corresponding to each 5′

-end portion of said first and second primers used in steps (b) and (e), respectively, under conditions in which each primer anneals to the 3′

- and 5′

-end sequences of each second cDNA strand, respectively, whereby amplification products of said second cDNA strands are generated, and

(g) comparing the presence or level of individual amplification products in said first and second populations of amplification products obtained from step (f).

47. A method for rapidly amplifying a target cDNA fragment comprising a cDNA region corresponding to the 3′

-end region of an mRNA, wherein said method comprises reverse transcribing said mRNA and performing an amplification reaction using primers, characterized in that at least one primer has the same structure as the primer of claim 1.

48. The method according to claim 47, wherein said method is performed using two stage amplifications, which comprises:

(a) contacting mRNAs with a first primer of claim 1, in which the 3′

-end portion of said primer comprises a hybridizing nucleotide sequence substantially complementary to poly A tails of said mRNAs to hybridize therewith, under conditions sufficient for template driven enzymatic deoxyribonucleic acid synthesis to occur;

(b) reverse transcribing said mRNAs to which said first primer hybridizes to produce a population of first cDNA strands that are complementary to said mRNAs to which said first primer hybridizes;

(c) performing a first-stage amplification of said first cDNA strands at a first annealing temperature comprising at least one cycle of primer annealing, primer extending and denaturing, using a second primer of claim 1 having at its 3′

-end portion a gene-specific hybridizing nucleotide sequence substantially complementary to a site in one of said first cDNA strands to hybridize therewith, under conditions in which said second primer anneals to a gene-specific site on one of said first cDNA strands, whereby a gene-specific second cDNA strand is generated; and

(d) performing a second-stage amplification of said gene-specific second cDNA strand generated from step (c) at a second annealing temperature, which is high stringent conditions, comprising at least two cycles of primer annealing, primer extending and denaturing, using the same first and second primers as used in steps (a) and (c), respectively, or a primer pair each comprising a pre-selected arbitrary nucleotide sequence corresponding to each 5′

-end portion of said first and second primers used in steps (a) and (c), respectively, under conditions in which each primer anneals to the 3′

- and 5′

-end sequences of a gene-specific second cDNA strand, respectively, whereby an amplification product of a gene-specific cDNA strand is generated.

49. A method for rapidly amplifying a target DNA fragment comprising a cDNA region corresponding to the 5′

-end region of an mRNA, wherein said method comprises reverse transcribing said mRNA and performing an amplification reaction using primers, characterized in that at least one primer has the same structure as the primer of claim 1.

50. The method according to claim 49, wherein said method is performed using two stage amplifications, which comprises:

(a) contacting mRNAs with an oligonucleotide dT primer or random primer as a cDNA synthesis primer under conditions sufficient for template driven enzymatic deoxyribonucleic acid synthesis to occur, in which said cDNA synthesis primer comprises a hybridizing nucleotide sequence substantially complementary to a region of an mRNA to hybridize therewith;

(b) reverse transcribing said mRNAs, using a reverse transcriptase, to which said cDNA synthesis primer hybridizes to produce a population of first cDNA strands that are complementary to said mRNAs to which said cDNA synthesis primer hybridizes, whereby mRNA-cDNA intermediates are generated;

(c) permitting cytosine residues to be tailed at the 3′

-ends of said first cDNA strands in the form of said mRNA-cDNA intermediates by the terminal transferase reaction of reverse transcriptase;

(d) contacting the cytosine tails at the 3′

-ends of said first cDNA strands generated from step (c) with an oligonucleotide which comprises a 3′

-end portion and a 5′

-end portion separated by a group of universal base or non-discriminatory base analog, wherein the 3′

-end portion comprises at least three guanine residues at its 3′

-end to hybridize with said cytosine tails at the 3′

-ends of said first cDNA strands and the 5′

-end portion comprises a pre-selected arbitrary nucleotide sequence, under conditions in which said 3-end portion of said oligonucleotide is hybridized to said cytosine tails;

(e) extending the tailed 3′

-ends of said first cDNA strands to generate an additional sequence complementary to said oligonucleotide using reverse transcriptase, in which said oligonucleotide serves as a template in the extension reaction, whereby full-length first cDNA strands are extended;

(f) performing a first-stage amplification of said full-length first cDNA strands obtained from step (e) at a first annealing temperature, which comprises the steps of;

(i) at least one cycle of primer annealing, primer extending and denaturing using a first primer comprising a nucleotide sequence substantially complementary to the 3′

-end sequences of said full-length first cDNA strands under conditions in which said first primer anneals to said full-length first cDNA strands, under conditions in which said first primer anneals to the 3′

- ends of said full-length first cDNA strands, whereby full-length second cDNA strands are generated;

(ii) at least one cycle of primer annealing, primer extending and denaturing using a second primer of claim 1 having at its 3′

-end portion a gene-specific hybridizing sequence substantially complementary to a region on one of said full-length second cDNA strands to hybridize therewith, under conditions in which said second primer anneals to a gene-specific site on one of said full-length second cDNA strands, whereby a gene-specific cDNA strand is generated; and

(g) performing a second-stage amplification of said gene-specific cDNA strand at a second annealing temperature, which is high stringent conditions, comprising at least two cycles of primer annealing, primer extending and denaturing, using the same first and second primers as used in steps (f)-(i) and (f)-(ii), respectively, or a primer pair each comprising a nucleotide sequence corresponding to each 5′

-end portion of said first and second primers as used in steps (f)-(i) and (f)-(ii), respectively, under conditions in which each primer anneals to the 3′

- and 5′

-end sequences of a gene-specific cDNA strand, respectively, whereby an amplification product of a gene-specific cDNA strand is generated.

51. A method for amplifying a population of full-length double-stranded cDNAs complementary to mRNAs, wherein said method comprises reverse transcribing said mRNA and performing an amplification reaction using primers, characterized in that at least one primer has the same structure as the primer of claim 1.

52. The method according to claim 51, wherein said method comprises:

(a) contacting said mRNAs with a first primer of claim 1, in which the 3′

-end portion of said first primer has a hybridizing nucleotide sequence substantially complementary to poly A tails of said mRNAs to hybridize therewith, under conditions sufficient for template driven enzymatic deoxyribonucleic acid synthesis to occur;

(b) reverse transcribing said mRNAs, using a reverse transcriptase, to which said first primer hybridizes to produce said population of first cDNA strands that are complementary to said mRNAs to which said primer hybridizes, whereby mRNA-cDNA intermediates are generated;

(c) permitting cytosine residues to be tailed at the 3′

-ends of said first cDNA strands in the form of said mRNA-cDNA intermediates by the terminal transferase reaction of reverse transcriptase;

(d) contacting the cytosine tails at the 3′

-ends of said first cDNA strands generated from step (c) with an oligonucleotide which comprises a 3′

-end portion and a 5′

-end portion separated by a group of universal base or non-discriminatory base analog, wherein the 3′

-end portion comprises at least three guanine residues at its 3′

-end to hybridize with said cytosine tails at the 3′

-ends of said first cDNA strands and the 5′

-end portion comprises a pre-selected arbitrary nucleotide sequence, under conditions in which said 3-end portion of said oligonucleotide is hybridized to said cytosine tails;

(e) extending the tailed 3′

-ends of said first cDNA strands to generate an additional sequence complementary to said oligonucleotide using reverse transcriptase, in which said oligonucleotide serves as a template in the extension reaction, whereby full-length first cDNA strands are extended; and

(f) performing an amplification of said full-length first cDNA strands generated from step (e) comprising at least two cycles of primer annealing, primer extending and denaturing, 4 0 using a primer pair each comprising a nucleotide sequence corresponding to the same first primer and oligonucleotide as used in steps (a) and (d), respectively, or a primer pair each comprising a nucleotide sequence corresponding to each 5′

-end portion of said first primer and oligonucleotide used in steps (a) and (d), respectively, under conditions in which each primer anneals to the 3′

- and 5′

-end sequences of said full-length first cDNA strands, respectively, whereby amplification products of full-length cDNA strands complementary to said mRNAs are generated.

53. A method for amplifying 5′

-enriched double-stranded cDNAs complementary to mRNAs, wherein said method comprises comprising reverse transcribing said mRNAs and performing an amplification reaction using primers, characterized in that at least one primer has the same structure as the primer of claim 1.

54. The method according to claim 53, wherein said method comprises:

(a) contacting said mRNAs with a first primer of claim 1 under conditions sufficient for template driven enzymatic deoxyribonucleic acid synthesis to occur, wherein the 3′

-end portion of said first primer has at least six random nucleotide sequences;

(b) performing the steps (b)-(e) of claim 52, whereby 5′

-enriched first cDNA strands are extended;

(c) performing an amplification of said 5′

-enriched first cDNA strands generated from step (b) comprising at least two cycles of primer annealing, primer extending and denaturing, using a primer pair each comprising a nucleotide sequence corresponding to each 5′

-end portion of said primer and oligonucleotide used in steps (a) and (b), respectively, under conditions in which each primer anneals to the 3′

- and 5′

-end sequences of said 5′

-enriched first cDNA strands, respectively, whereby amplification products of 5′

-enriched cDNA strands are generated.

55. A method for amplifying more than one target nucleotide sequence simultaneously using more than one pair of primers in the same reaction, wherein said method comprises performing an amplification reaction using primers, characterized in that at least one primer has the same structure as the primer of claim 1.

56. The method according to claim 55, wherein said method is performed using two stage amplifications, which comprises:

(a) performing a first-stage amplification of more than one target nucleotide sequence at a first annealing temperature comprising at least two cycles of primer annealing, primer extending and denaturing, using the primer pairs of claim 1 in which its 3′

end portion each of each primer pair has a hybridizing nucleotide sequence substantially complementary to a region of said target nucleic acid sequence to hybridize therewith, under conditions in which each of said primer pairs anneals to its target nucleotide sequence, whereby the amplification products of target nucleotide sequences are generated; and

(b) performing a second-stage amplification of said amplification products generated from step (a) at a second annealing temperature, which is high stringent conditions, comprising at least one cycle of primer annealing, primer extending and denaturing, using the same primer pairs as used in step (a) or primer pairs each comprising a pre-selected arbitrary nucleotide sequence corresponding to each 5′

-end portion of said primer pairs used in step (a), under conditions in which each of each primer pair anneals to the 3′

- and 5′

-end sequences of said amplification products generated from step (a), respectively, whereby said amplification products are re-amplified in the same reaction.

57. A method for producing a DNA fingerprint of gDNA, wherein said method comprises performing an amplification reaction using primers, characterized in that at least one primer has the same structure as the primer of claim 1.

58. The method according to claim 57, wherein said method is performed using two stage amplifications, which comprises:

(a) performing a first-stage amplification of said DNA fingerprint, which is a set of discrete DNA segments characteristic of genome, from said gDNA at a first annealing temperature comprising at least two cycles of primer annealing, primer extending and denaturing, using the primer or the primer pair of claim 1, wherein each primer has at its 3′

-end portion an arbitrary nucleotide sequence substantially complementary to sites on said gDNA to hybridize therewith, under conditions in which said primer or said primer pair anneals to said gDNA, whereby said set of discrete DNA segments characterized as a DNA fingerprint is produced; and

(b) performing a second-stage amplification of said set of discrete DNA segments generated from step (a) at a second annealing temperature, which is high stringent conditions, comprising at least one cycle of primer annealing, primer extending and denaturing, using the same primer or primer pair as used in step (a) or a primer or a primer pair each comprising a nucleotide sequence corresponding to each 5′

-end portion of said primer or primer pair used in step (a), under conditions in which said primer or each of said primer pair anneals to the 3′

- and 5′

-end sequences of said set of discrete DNA segments generated from step (a), respectively, whereby said set of discrete DNA segments is re-amplified.

59. A method for producing a RNA fingerprint of an mRNA sample, wherein said method comprises reverse transcribing and performing an amplification reaction using primers, characterized in that at least one primer has the same structure as the primer of claim 1.

60. The method according to claim 59, wherein said method is performed using two stage amplifications, which comprises:

(a) contacting said mRNA sample with a first primer of claim 1, in which said first primer has a hybridizing nucleotide sequence substantially complementary to poly A tails of said mRNA sample to hybridize therewith, under conditions sufficient for template driven enzymatic deoxyribonucleic acid synthesis to occur;

(b) reverse transcribing said mRNA sample to which said first primer hybridizes to produce a population of first cDNA strands that are complementary to said mRNA sample to which said first primer hybridizes;

(c) performing a first-stage amplification of said population of first cDNA strands generated from step (b) at a first annealing temperature comprising at least one cycle of primer annealing, primer extending and denaturing, using a second primer or primer pair of claim 1, wherein each primer has at its 3′

-end portion an arbitrary nucleotide sequence substantially complementary to sites on said first cDNA strands to hybridize therewith, under conditions in which said primer or primer pair anneals to said mRNA sample, whereby a set of discrete cDNA segments characterized as a RNA fingerprint is produced; and

(d) performing a second stage amplification of said set of discrete cDNA segments generated from step (c) at a second annealing temperature which is high stringent conditions, comprising at least one cycle of primer annealing, primer extending and denaturing, using the same primer or primer pair as used in step (c) or a primer or primer pair each comprising a nucleotide sequence corresponding to each 5′

-end portion of said primer or primer pair used in step (c), under conditions in which said primer or each of said primer pair anneals to the 3′

- and 5′

-end sequences of said set of discrete cDNA segments generated from step (c), respectively, whereby said set of discrete cDNA segments is re-amplified.

61. A method for identifying conserved homology segments in a multigene family from an mRNA sample, wherein said method comprises reverse transcribing and performing an amplification reaction using primers, characterized in that at least one primer has the same structure as the primer of claim 1.

62. The method according to claim 61, wherein said method is performed using two stage amplifications, which comprises:

(a) contacting said mRNA sample with a first primer of claim 1, in which said first primer has a hybridizing nucleotide sequence substantially complementary to poly A tails of said mRNA sample to hybridize therewith, under conditions sufficient for template driven enzymatic deoxyribonucleic acid synthesis to occur;

(b) reverse transcribing said mRNA sample to which said first primer hybridizes to produce a population of first cDNA strands that are complementary to said mRNA sample to which said first primer hybridizes;

(c) performing a first-stage amplification of said population of first cDNA strands generated from step (b) at a first annealing temperature comprising at least one cycle of claim 1 having at its 3′

end portion a hybridizing sequence substantially complementary to a consensus sequence or a degenerate sequence encoding amino acid sequence of a conserved homology segment on said first cDNA strands to hybridize therewith, under conditions in which said second primer anneals to said consensus sequence or degenerate sequence of first cDNA strands, whereby 3′

-end cDNA segments having said consensus sequence or degenerate sequence are generated; and

(d) performing a second stage amplification of said 3′

-end cDNA segments generated from step (c) at a second annealing temperature which is high stringent conditions, comprising at least two cycles of primer annealing, primer extending and denaturing, using the same first and second primers as used in steps (a) and (c) or a primer pair each comprising a nucleotide sequence corresponding to each 5′

-end portion of said first and second primers used in steps (a) and (c), respectively, under conditions in which each primer anneals to the 3′

- and 5′

-end sequences of said 3′

-end cDNA segments, respectively, whereby said 3′

-end conserved homology cDNA segments are amplified.

63. The method according to claim 61, wherein said method is performed using two stage 10 amplifications, which comprises:

(a) performing steps of (a)-(e) of claim 52, whereby full-length cDNA strands are generated;

(b) performing a first-stage amplification of said full-length first cDNA strands obtained from step (a) at a first annealing temperature, which comprises the steps of;

(i) at least one cycle of primer annealing, primer extending and denaturing using a first primer comprising a nucleotide sequence substantially complementary to the 3′

-end sequences of said full-length first cDNA strands under conditions in which said first primer anneals to said full-length first cDNA strands, under conditions in which said first primer anneals to the 3′

- ends of said full-length first cDNA strands, whereby full-length second cDNA strands are generated; and

(ii) at least one cycle of primer annealing, primer extending and denaturing using a second primer of claim 1 having at its 3′

end portion a hybridizing sequence substantially complementary to a consensus sequence or a degenerate sequence encoding amino acid sequence of a conserved homology segment on said full-length second cDNA strands to hybridize therewith, under conditions in which said second primer anneals to said consensus sequence or degenerate sequence of full-length second cDNA strands, whereby 5′

-end cDNA segments having said consensus sequence or degenerate sequence are generated; and

(c) performing a second stage amplification of said 5′

-end cDNA segments generated from step (b) at a second annealing temperature which is high stringent conditions, comprising at least two cycles of primer annealing, primer extending and denaturing, using the same first and second primers as used in steps (b)-(i) and (b)-(ii), respectively, or a primer pair each comprising a nucleotide sequence corresponding to each 5′

-end portion of said first and second primers used in steps (b)-(i) and (b)-(ii), respectively, under conditions in which each primer anneals to the 3′

- and 5′

-end sequences of said 5′

-end cDNA segments, respectively, whereby said 5′

-end conserved homology cDNA segments are amplified.

64. A method for identifying conserved homology segments in a multigene family from gDNA, wherein said method comprises performing an amplification reaction using primers, characterized in that at least one primer has the same structure as the primer of claim 1.

65. The method according to claim 64, wherein said method is performed using two stage amplifications, which comprises:

(a) performing a first-stage amplification of said conserved homology segments from said gDNA at a first annealing temperature comprising at least two cycles of primer annealing, primer extending and denaturing, using the primer or the primer pair of claim 1, wherein each primer has at its 3′

end portion a hybridizing sequence substantially complementary to a consensus sequence or a degenerate sequence encoding amino acid sequence of a conserved homology segment on said gDNA to hybridize therewith, under conditions in which said primer or said primer pair anneals to said consensus sequence or degenerate sequence of gDNA, whereby genomic DNA segments having said consensus sequence or degenerate sequence are generated; and

(b) performing a second-stage amplification of said genomic DNA segments generated from step (a) at a second annealing temperature, which is high stringent conditions, comprising at least one cycle of primer annealing, primer extending and denaturing, using the same primer or primer pair as used in step (a) or a primer or a primer pair each comprising a nucleotide sequence corresponding to each 5′

-end portion of said primer or primer pair used in step (a), under conditions in which said primer or each of said primer pair anneals to the 3′

- and 5′

-end sequences of said genomic DNA segments generated from step (a), respectively, whereby said conserved homology genomic segments are amplified.

66. A method for identifying a nucleotide variation in a target nucleic acid, wherein said method comprises performing an amplification reaction using primers, characterized in that at least one primer has the same structure as the primer of claim 1.

67. The method according to claim 66, wherein said method is performed using two stage amplifications, which comprises:

(a) performing a first-stage amplification to produce a first DNA strand complementary to said target nucleic acid including said nucleotide variation at a first annealing temperature comprising at least one cycle of primer annealing, primer extending and denaturing, using a first primer of claim 1 having at its 3′

-end portion a hybridizing sequence substantially complementary to a pre-selected sequence at a first site of said target nucleic acid to hybridize therewith, wherein each of said first primer and said first site comprises an interrogation position corresponding to said nucleotide variation, whereby said first DNA strand complementary to said target nucleic acid including said nucleotide variation is generated when said interrogation position is occupied by the complementary nucleotide of said first primer to its corresponding nucleotide of said first site; and

(b) performing a second-stage amplification of said first DNA strand generated from step (a) at a second annealing temperature, which is high stringent conditions, comprising the steps;

(i) at least one cycle of primer annealing, primer extending and denaturing using a second primer of claim 1 having at its 3′

-end portion a hybridizing sequence substantially complementary to a pre-selected sequence at a second site of said target nucleic acid to hybridize therewith under conditions in which said second primer anneals to said second site of the target nucleic acid, whereby a second DNA strand complementary to said first DNA strand including said nucleotide variation is generated; and

(ii) at least one cycle of primer annealing, primer extending and denaturing using the same first and second primers as used in steps (a) and (b)-(i) or a primer pair each having a hybridizing sequence complementary or corresponding to the 3′

- and 5′

-ends of said second DNA strand generated from step (b)-(i) to hybridize therewith, under conditions in which each primer anneals to the 3′

- and 5′

-end sequences of said second DNA strand, respectively, whereby said second DNA strand which comprises said first and second sites of said target nucleic acid at its 3′

-and 5′

-ends is amplified so that a short target nucleotide segment corresponding to said second DNA strand containing said nucleotide variation is generated.

68. The method according to claim 66, wherein said method is performed using two individual amplifications of a first and a second amplifications in which said second amplification is performed using two stage amplifications, which comprises:

(a) performing said first amplification to produce a short DNA strand fragment containing said nucleotide variation between its ends comprising at least two cycles of primer annealing, primer extending and denaturing, using a primer pair each primer comprising a hybridizing sequence substantially complementary to a pre-selected sequence at a site of said target nucleic acid under conditions that said nucleotide variation is positioned between said pre-selected sequences, in which at least one primer of said primer set is the primer of claim 1 having at its 3′

-end portion said hybridizing sequence, whereby said short DNA strand fragment containing said nucleotide variation between its ends is amplified;

(b) performing a first-stage amplification of said second amplification to produce a first DNA strand complementary to said short DNA strand fragment including said nucleotide variation at a first annealing temperature comprising at least one cycle of primer annealing, primer extending and denaturing, using a first primer of claim lhaving at its 3′

-end portion a hybridizing sequence substantially complementary to a pre-selected sequence at a first site of said target nucleic acid to hybridize therewith, wherein each of said first primer and said first site comprises an interrogation position corresponding to said nucleotide variation, whereby said first DNA strand complementary to said target nucleic acid including said nucleotide variation is generated when said interrogation position is occupied by the complementary nucleotide of said first primer to its corresponding nucleotide of said first site; and

(c) performing a second-stage amplification of said second amplification of said first DNA strand generated from step (a) at a second annealing temperature, which is high stringent conditions, comprising at least one cycle of primer annealing, primer extending and denaturing using a primer pair in which amongst said primer pair one is the same as said primer of claim 1 used in step (a) the other is the same as said first primer used in step (b), or a primer pair each having a hybridizing sequence complementary or corresponding to the 3′

- and 5′

-ends of said first DNA strand generated from step (b) to hybridize therewith, under conditions in which each primer anneals to the 3′

- and 5′

-end sequences of said first DNA strand, respectively, whereby said first DNA strand is amplified so that a short target nucleotide segment corresponding to said first DNA strand containing said nucleotide variation is generated.

69. A method for mutagenesis in a target nucleic acid, comprising performing an amplification reaction using primers, characterized in that at least one primer has the same structure as the primer of claim 1.

70. The method according to claim 69, wherein said mutagenesis is site-directed and uses two stages amplifications, comprising the steps of:

(a) performing a first-stage amplification of said target nucleic acid sequence at a first annealing temperature comprising at least two cycles of primer annealing, primer extending and denaturing, using a primer pair of claim 1 each having at its 3′

end portion a hybridizing sequence substantially complementary to a region of said target nucleic acid sequence to hybridize therewith, wherein said hybridizing sequence has at least one mismatch nucleotide to generate site-directed mutation, under conditions in which said primer or primer pair anneals to its target nucleotide sequence, whereby an amplification product containing site-directed mutation site is generated; and

(b) performing a second-stage amplification of said amplification product generated from step (a) at a second annealing temperature, which is high stringent conditions, comprising at least one cycle of primer annealing, primer extending and denaturing, using the same primers as used in step (a) or a primer pair each comprising a pre-selected arbitrary nucleotide sequence corresponding to each 5′

-end portion of said primers used in step (a), under conditions in which each primer anneals to the 3′

- and 5′

-ends of said amplification product, respectively, whereby said amplification product containing site-directed mutation site is re-amplified.

71. A kit for nucleic acid amplification, which comprises the annealing control primer or annealing control primer set described in claim 40.

72. A kit for selective amplification of a target nucleic acid sequence from DNA, which comprises the annealing control primer or annealing control primer set described in claim 42.

73. A kit for selective amplification of a target nucleic acid sequence from mRNA, which comprises the annealing control primer or annealing control primer set described in claim 44.

74. A kit for detecting DNA complementary to differentially expressed mRNA, which comprises the annealing control primer or annealing control primer set described in claim 46.

75. A kit for rapidly amplifying a target cDNA fragment comprising a cDNA region corresponding to the 3′

-end region of an mRNA, which comprises the annealing control primer or annealing control primer set described in claim 48.

76. A kit for rapidly amplifying a target cDNA fragment comprising a cDNA region corresponding to the 5′

-end region of an mRNA, which comprises the annealing control primer or annealing control primer set described in claim 50.

77. A kit for amplifying a population of full-length double-stranded cDNAs complementary to mRNAs, which comprises the annealing control primer or annealing control primer set described in claim 52.

78. A kit for amplifying 5′

-enriched double-stranded cDNAs complementary to the 5′

-end regions of mRNAs, which comprises the annealing control primer or annealing control primer set described in claim 54.

79. A kit for amplifying more than one target nucleotide sequence simultaneously, which comprises the annealing control primer or annealing control primer set described in claim 56.

80. A kit for producing a DNA fingerprint by use of gDNA, which comprises the annealing control primer or annealing control primer set described in claim 58.

81. A kit for producing a RNA fingerprint by use of mRNA, which comprises the annealing control primer or annealing control primer set described in claim 60.

82. A kit for identifying a conserved homology segment in a multigene family by use of mRNA, which comprises the annealing control primer or annealing control primer set described in claim 62 or 63.

83. A kit for identifying a conserved homology segment in a multigene family by use of gDNA, which comprises the annealing control primer or annealing control primer set described in claim 65.

84. A kit for identifying a nucleotide variation in a target nucleic acid, which comprises the annealing control primer or annealing control primer set described in claim 67 or 68.

85. A kit for mutagenesis in a target nucleic acid, which comprises the annealing control primer or annealing control primer set described in claim 70.