Patent EA-004327-B1 (EA)
Title: Method for amplifying nucleic acid sequence
Inventor: MUKAI HIROYUKI
IPC: C12Q1/68
Filed: 2000-03-14
Abstract: 1. A method for amplifying a nucleotide sequence, characterized in that the method comprises: (a) treating a nucleic acid as a template with at least one primer that is substantially complementary to the nucleotide sequence of the nucleic acid and a DNA polymerase to synthesize a primer-extended strand that is complementary to the template, wherein the primer is a chimeric oligonucleotide primer containing a deoxyribonucleotide and a ribonucleotide, the ribonucleotide being positioned at the 3'-terminus or on the 3'-terminal side of the primer for cleavage with an endonuclease; (b) cleaving the primer-extended strand of a double-stranded nucleic acid obtained in step (a) with the endonuclease at a site that contains the ribonucleotide; and (c) extending a nucleotide sequence that is complementary to the template using a DNA polymerase having a strand displacement activity from the 3'-terminus of the primer portion of the double-stranded nucleic acid in which the primer-extended strand is cleaved obtained in step (b) to effect a strand displacement. 2. The method according to claim 1, wherein step (b) and step (c) are sequentially repeated. 3. A method for amplifying a nucleotide sequence using at least two primers, characterized in that the method comprises: (a) treating a nucleic acid as a template with at least one primer that is substantially complementary to the nucleotide sequence of the nucleic acid and a DNA polymerase to synthesize a primer-extended strand that is complementary to the template, wherein the primer is a chimeric oligonucleotide primer containing a deoxyribonucleotide and a ribonucleotide, the ribonucleotide being positioned at the 3'-terminus or on the 3'-terminal side of the primer for cleavage with an endonuclease; (b) cleaving the primer-extended strand of a double-stranded nucleic acid obtained in step (a) with the endonuclease at a site that contains the ribonucleotide; (c) extending a nucleotide sequence that is complementary to the template using a DNA polymerase having a strand displacement activity from the 3'-terminus of the primer portion of the double-stranded nucleic acid in which the primer-extended strand is cleaved obtained in step (b) to effect a strand displacement, wherein a double-stranded nucleic acid containing a regenerated primer-extended strand is reused in step (b); (d) treating a released displaced strand obtained in step (c) as a template with at least one primer that is different from that used in step (a) and a DNA polymerase to synthesize a primer-extended strand that is complementary to the displaced strand, wherein the primer that is different from that used in step (a) is a chimeric oligonucleotide primer that is substantially complementary to the nucleotide sequence of the displaced strand and contains a deoxyribonucleotide and a ribonucleotide, the ribonucleotide being positioned at the 3'-terminus or on the 3'-terminal side of the primer for cleavage with an endonuclease; (e) cleaving the primer-extended strand of a double-stranded nucleic acid obtained in step (d) with the endonuclease at a site that contains the ribonucleotide; and (f) extending a nucleotide sequence that is complementary to the template using a DNA polymerase having a strand displacement activity from the 3'-terminus of the primer portion of the double-stranded nucleic acid in which the primer-extended strand is cleaved obtained in step (e) to effect a strand displacement, wherein a double-stranded nucleic acid containing a regenerated primer-extended strand is reused in step (e). 4. The method according to any one of claims 1 to 3, wherein the respective steps are conducted under isothermal conditions. 5. The method according to any one of claims 1 to 4, wherein one DNA polymerase having a strand displacement activity is used. 6. The method according to any one of claims 1 to 5, wherein the DNA polymerase is selected from the group consisting of Klenow fragment of DNA polymerase I from Escherichia coli, Bst DNA polymerase lacking 5'->3' exonuclease from Bacillus stearothermophilus and Bca DNA polymerase lacking 5'->3' exonuclease from Bacillus caldotenax. 7. The method according to any one of claims 1 to 6, wherein the endonuclease is an endoribonuclease. 8. The method according to claim 7, wherein the endoribonuclease is RNase H. 9. The method according to any one of claims 1 to 8, wherein the chimeric oligonucleotide primer contains two or more successive ribonucleotide residues. 10. The method according to any one of claims 1 to 9, wherein the chimeric oligonucleotide primer contains one or more modified ribonucleotide. 11. The method according to claim 10, wherein the chimeric oligonucleotide primer contains an (alpha-S) ribonucleotide in which the oxygen atom bound to the phosphorous atom at the alpha -position of the ribonucleotide is replaced by a sulfur atom. 12. The method according to any one of claims 1 to 11, which is conducted in a buffer that contains a buffering component selected from the group consisting of Tricine, a phosphate and tris. 13. A method for amplifying a nucleotide sequence, characterized in that the method comprises: (a) preparing a reaction mixture by mixing a nucleic acid as a template, a deoxyribonucleotide triphosphate, a DNA polymerase having a strand displacement activity, at least one primer and an endonuclease that cleaves an extended strand generated from the primer, wherein the primer is a chimeric oligonucleotide primer that is substantially complementary to the nucleotide sequence of the nucleic acid as the template and contains a deoxyribonucleotide and a ribonucleotide, the ribonucleotide being positioned at the 3'-terminus or on the 3'-terminal side of the primer for cleavage with the endonuclease; and (b) incubating the reaction mixture for a sufficient time to generate a reaction product. 14. The method according to claim 13, wherein the reaction mixture is incubated under isothermal conditions. 15. The method according to claim 13 or 14, wherein the reaction mixture further contains a chimeric oligonucleotide primer having a sequence substantially homologous to the nucleotide sequence of the nucleic acid as the template. 16. The method according to any one of claims 13 to 15, wherein the DNA polymerase is selected from the group consisting of Klenow fragment of DNA polymerase I from Escherichia coli, Bst DNA polymerase lacking 5'->3' exonuclease from Bacillus stearothermophilus and Bca DNA polymerase lacking 5'->3' exonuclease from Bacillus caldotenax. 17. The method according to any one of claims 13 to 16, wherein the endonuclease is an endoribonuclease. 18. The method according to claim 17, wherein the endoribonuclease is RNase H. 19. The method according to any one of claims 13 to 18, wherein the chimeric oligonucleotide primer contains two or more successive ribonucleotide residues. 20. The method according to any one of claims 13 to 19, wherein the chimeric oligonucleotide primer contains one or more modified ribonucleotide. 21. The method according to claim 20, wherein the chimeric oligonucleotide primer contains an (alpha -S) ribonucleotide in which the oxygen atom bound to the phosphorous atom at the alpha -position of the ribonucleotide is replaced by a sulfur atom. 22. The method according to any one of claims 13 to 21, which is conducted in a buffer that contains a buffering component selected from the group consisting of Tricine, a phosphate and tris. 23. The method according any one of claims 1 to 22, wherein the nucleic acid as the template is a single-stranded DNA or a double-stranded DNA. 24. The method according to claim 23, which is conducted after converting a double-stranded DNA as the template into single-stranded DNAs. 25. The method according to claim 23 or 24, wherein the nucleic acid as the template is a cDNA obtained from an RNA by a reverse transcription reaction. 26. The method according to claim 25, which is conducted after synthesizing a cDNA by a reverse transcription reaction using an RNA as a template. 27. The method according to claim 26, wherein a primer selected from the group consisting of an oligo-dT primer, a random primer and a specific primer is used as a primer for the reverse transcription reaction. 28. The method according to claim 26 or 27, wherein a chimeric oligonucleotide primer is used as a primer for the reverse transcription reaction. 29. The method according to any one of claims 26 to 28, wherein a DNA polymerase having a reverse transcriptase activity is used as a reverse transcriptase. 30. The method according to any one of claims 26 to 29, wherein the reverse transcription reaction and the synthesis of the extended strand that is complementary to the template are conducted using one DNA polymerase having a reverse transcriptase activity and a strand displacement activity. 31. The method according to claim 28, wherein the DNA polymerase is Bst DNA polymerase lacking 5'->3' exonuclease from Bacillus stearothermophilus or Bca DNA polymerase lacking 5'->3' exonuclease from Bacillus caldotenax. 32. The method according to any one of claims 25 to 31, wherein the RNA as the template in the reverse transcription reaction is an RNA amplified by a nucleic acid amplification reaction. 33. The method according to claim 32, which is conducted after synthesizing an amplified RNA fragment by a nucleic acid amplification reaction using an RNA as a template. 34. The method according to claim 32 or 33, wherein the nucleic acid amplification reaction is selected from the group consisting of the transcription-based amplification system (TAS) method, the self-sustained sequence replication (3SR) method, the nucleic acid sequence-based amplification (NASBA) method, the transcription-mediated amplification (TMA) method and the Qbeta replicase method. 35. The method according to claim 23 or 24, wherein the nucleic acid as the template is a DNA obtained by a nucleic acid amplification reaction. 36. The