Patent EA-005141-B1 (EA)
Title: Method of detecting nucleotide polymorphism
Inventor: SAGAWA HIROAKI
IPC: C12Q1/68
Filed: 2002-02-14
Abstract: 1. A method for detecting the presence of a base substitution at a specific base in a target nucleic acid, the method comprising: (1) mixing a sample containing a target nucleic acid with a Nucleotide, wherein the Nucleotide (A) is modified at the 3'-terminus such that extension from the terminus by a DNA polymerase does not occur; (B) has a base sequence capable of annealing to a region containing a specific base in the target nucleic acid; and (C) contains a sequence in which if there is a mismatch between the specific base and a base corresponding to the specific base in the Nucleotide in a complex composed of the Nucleotide and the target nucleic acid, the Nucleotide is not cleaved with a nuclease, and if there is no mismatch between the specific base and a base corresponding to the specific base in the Nucleotide, the Nucleotide is cleaved with a nuclease to generate a new 3'-terminus; (2) treating the mixture with the nuclease and the DNA polymerase; and (3) detecting the presence of cleavage of the Nucleotide with the nuclease. 2. The method according to claim 1, wherein the nuclease is a ribonuclease H, and the Nucleotide contains a ribonucleotide in the region containing the base corresponding to the specific base. 3. The method according to claim 1, wherein the nuclease is a restriction enzyme, and the Nucleotide contains a recognition sequence for the restriction enzyme in the region containing the base corresponding to the specific base. 4. A method for detecting the presence of a base substitution at a specific base in a target nucleic. acid, the method comprising: (1) mixing a sample containing a target nucleic acid with a Nucleotide, wherein the Nucleotide (A) is modified at the 3'-terminus such that extension from the terminus by a DNA polymerase does not occur; (B) has a base sequence capable of annealing to a region containing a specific base in the target nucleic acid; and (C) contains a sequence in which if there is no mismatch between the specific base and a base corresponding to the specific base in the Nucleotide in a complex composed of the Nucleotide and the target nucleic acid, the Nucleotide is not cleaved with a nuclease, and if there is a mismatch between the specific base and a base corresponding to the specific base in the Nucleotide, the Nucleotide is cleaved with a nuclease to generate a new 3'-terminus; (2) treating the mixture with the nuclease and the DNA polymerase; and (3) detecting the presence of cleavage of the Nucleotide with the nuclease. 5. The method according to claim 4, wherein the nuclease is a mismatch-specific nuclease. 6. The method according to claim 1 or 4, wherein the Nucleotide has a sequence in which if there is no base substitution in the target nucleic acid, a mismatch is not generated in the complex composed of the Nucleotide and the target nucleic acid. 7. The method according to claim 1 or 4, wherein the Nucleotide has a sequence in which if there is a base substitution in the target nucleic acid, a mismatch is not generated in the complex composed of the Nucleotide and the target nucleic acid. 8. The method according to claim 1 or 4, wherein the cleavage of the Nucleotide is detected based on the presence of an extension product generated by the action of the DNA polymerase. 9. The method according to claim 1 or 4, wherein the cleavage of the Nucleotide is detected based on the presence of a fragment of a 3' portion released from the Nucleotide generated by the action of the nuclease. 10. The method according to claim 1 or 4, wherein the cleavage of the Nucleotide is detected using a labeled compound attached to the Nucleotide. 11. The method according to claim 10, wherein the labeled compound is attached to the Nucleotide in a portion 3' to the cleavage site for the nuclease. 12. The method according to claim 10, wherein the labeled compound is attached to the Nucleotide in a portion 5' to the cleavage site for the nuclease. 13. The method according to claim 10, wherein the labeled compound attached to the Nucleotide is a fluorescent substance. 14. The method according to claim 13, wherein a substance capable of quenching fluorescence is further attached to the Nucleotide, and the fluorescence is emitted upon cleavage by the nuclease. 15. The method according to claim 13, wherein the cleavage of the Nucleotide is detected by a fluorescence polarization method. 16. The method according to claim 1 or 4, wherein the modification of the Nucleotide at the 3'-terminus is modification of the hydroxyl group at the 3-position of ribose. 17. The method according to claim 1 or 4, wherein the Nucleotide contains a nucleotide analog and/or a modified nucleotide. 18. The method according to claim 17, wherein the nucleotide analog is a deoxyriboinosine nucleotide or a deoxyribouracil nucleotide, and the modified ribonucleotide is an (alpha-S) ribonucleotide. 19. The method according to claim 1 or 4, further comprising a step of nucleic acid amplification in which an extension product generated by the action of the DNA polymerase is used as a template. 20. A method for analyzing a genotype of an allele; the method comprising detecting the presence of a base substitution at a specific base in a target nucleic acid according to the method defined by claim 19. 21. A Nucleotide used for detecting a base substitution at a specific base in a target nucleic acid, which (A) is modified at the 3'-terminus such that extension from the terminus by a DNA polymerase does not occur; (B) has a base sequence capable of annealing to a region containing a specific base in the target nucleic acid; and (C) contains a sequence in which if there is a mismatch between the specific base and a base corresponding to the specific base in the Nucleotide in a complex composed of the Nucleotide and the target nucleic acid, the Nucleotide is not cleaved with a nuclease, and if there is no mismatch between the specific base and a base corresponding to the specific base in the Nucleotide, the Nucleotide is cleaved with a nuclease to generate a new 3'-terminus. 22. The Nucleotide according to claim 21, which contains a ribonucleotide in the region containing the base corresponding to the specific base, wherein if there is no mismatch between the specific base and the base corresponding to the specific base in the Nucleotide in a complex composed of the Nucleotide and the target nucleic acid, the Nucleotide is cleaved with a ribonuclease H. 23. The Nucleotide according to claim 21, which contains a recognition sequence for a restriction enzyme in the region containing the base corresponding to the specific base, wherein if there is no mismatch between the specific base and the base corresponding to the specific base in the Nucleotide in a complex composed of the Nucleotide and the target nucleic acid, the Nucleotide is cleaved with the restriction enzyme. 24. A Nucleotide used in method for detecting according to claims 1 to 19, which (A) is modified at the 3'-terminus such that extension from the terminus by a DNA polymerase does not occur; (B) has a base sequence capable of annealing to a region containing a specific base in the target nucleic acid; and (C) contains a sequence in which if there is no mismatch between the specific base and a base corresponding to the specific base in the Nucleotide in a complex composed of the Nucleotide and the target nucleic acid, the Nucleotide is not cleaved with a nuclease, and if there is a mismatch between the specific base and a base corresponding to the specific base in the Nucleotide, the Nucleotide is cleaved with a nuclease to generate a new 3'-terminus. 25. The Nucleotide according to claim 24, wherein if there is a mismatch between the Nucleotide and the target nucleic acid in a complex composed of the Nucleotide and the target nucleic acid, the Nucleotide is cleaved with a mismatch-specific nuclease. 26. The Nucleotide according to claim 21 or 24, which has a sequence in which if there is no base substitution in the target nucleic acid, a mismatch is not generated in the complex composed of the Nucleotide and the target nucleic acid. 27. The Nucleotide according to claim 21 or 24, which has a sequence in which if there is a base substitution in the target nucleic acid, a mismatch is not generated in the complex composed of the Nucleotide and the target nucleic acid. 28. The Nucleotide according to claim 21 or 24, to which a labeled compound is attached. 29. The Nucleotide according to claim 28, wherein the labeled compound is attached to the Nucleotide in a portion 3' to the cleavage site for the nuclease. 30. The Nucleotide according to claim 28, wherein the labeled compound is attached to the Nucleotide in a portion 5' to the cleavage site for the nuclease. 31. The Nucleotide according to claim 28, wherein the labeled compound is a fluorescent substance. 32. The Nucleotide according to claim 31, to which a substance capable of quenching fluorescence is further attached, wherein the fluorescence is emitted upon cleavage by the nuclease or DNA extension subsequent to the cleavage. 33. The Nucleotide according to claim 21 or 24, wherein the modification of the Nucleotide at the 3'-terminus is modification of the hydroxyl group at the 3-position of ribose. 34. The Nucleotide according to claim 21 or 24, which contains a nucleotide analog and/or a modified nucleotide. 35. The Nucleotide according to claim 34, wherein the nucleotide analog is a deoxyriboinosine nucleotide or a deoxyribouracil nucleotide, and the modified ribonucleotide is an (alpha-S) ribonucleotide. 36. A kit used for detecting a base substitution in a target nucleic acid, which contains the Nucleotide defined by claim 21 or 24. 37. The kit according to claim 36, which contains a nuclease and/or a DNA polymerase. 38. The kit according to the claim 36, which further contains a reagent for detecting the presence of DNA extension. 39. The kit according to claim 36, which further contains