Method for amplifying nucleic acid and analysis of single-nucleotide polymorphism using the same

• US 20050277134A1
• Filed: 03/07/2005
• Published: 12/15/2005
• Est. Priority Date: 05/20/2004
• Status: Abandoned Application

• Current Assignee: Hitachi, Ltd.
• Original Assignee: Hitachi, Ltd.
• Inventors: Nakashima, Yukie, Okano, Kazunori
• Application Number:
  US11/072,510
• Publication Number:
  US 20050277134A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/185 incorporating bases where the precise position of the bases in the nucleic acid string is important

Abstract

A method amplifies a nucleic acid by preparing an oligonucleotide being capable of complementarily hybridizing with a specific region of a target nucleic acid containing at least one mutation site, the oligonucleotide having at least one sequence being non-complementary to any of possible sequences of the at least one mutation site, subjecting the oligonucleotide to hybridization with the target nucleic acid, and carrying out a complementary-strand synthesis. A single-nucleotide polymorphism is analyzed using this method.

Claims

1. A method for amplifying a nucleic acid comprising:

preparing an oligonucleotide being capable of complementarily hybridizing with a specific region of a target nucleic acid containing at least one mutation site, the oligonucleotide having at least one non-complementary sequence being not complementary to any of possible sequences of the at least one mutation site;

subjecting the oligonucleotide to hybridization with the target nucleic acid; and

carrying out a complementary-strand synthesis.

2. The method according to claim 1, wherein the oligonucleotide is allowed to hybridize with the target nucleic acid at temperatures of 45°

C. to 55°

C.

3. The method according to claim 1, wherein the oligonucleotide is allowed to hybridize with the target nucleic acid at temperatures of 47°

C. to 52°

C.

4. The method according to claim 1, wherein the non-complementary sequence is positioned at a third to fifteenth base from the 3′

end of the oligonucleotide.

5. The method according to claim 1, wherein the non-complementary sequence is a spacer or a base being not complementary to any of possible sequences of the at least one mutation site.

6. The method according to claim 1, wherein a region of the oligonucleotide capable of hybridizing with the target nucleic acid comprises 17 to 28 bases in length.

7. A method for analyzing a single-nucleotide polymorphism using an amplified product by the method of claim 1.

8. The method according to claim 7, which comprises:

typing a single-nucleotide polymorphism in the target nucleic acid other than the at least one mutation by the analysis of the amount of the amplified product,

wherein a base at the 3′

end or a second base from the 3′

end of the oligonucleotide is so designed as to correspond to the single-nucleotide polymorphism.

9. The method according to claim 8, wherein the typing comprises:

converting pyrophosphate into ATP, the pyrophosphate being generateed as a result of the complementary-strand synthesis;

carrying out a luminous reaction with the use of the resulting ATP and one or more enzymes; and

analyzing the amounts of an amplified product based on the quantity of light emitted as a result of the luminous reaction to thereby type the single-nucleotide polymorphism.

10. The method according to claim 7, which comprises typing a single-nucleotide polymorphism in the target nucleic acid other than the at least one mutation with the use of the amplified product as a template.

11. The method according to claim 10 further comprising:

subjecting an oligonucleotide probe to hybridization with the amplified product, the oligonucleotide probe being so designed as to have a corresponding base at the 3′

end or at a second base from the 3′

end, the base corresponding to the single-nucleotide polymorphism site;

carrying out a complementary-strand synthesis to yield an amplified product; and

typing the single-nucleotide polymorphism by the analysis of the amount of the amplified product.

12. The method according to claim 11, wherein the typing comprises:

converting pyrophosphate into ATP, the pyrophosphate being generateed as a result of the complementary-strand synthesis;

carrying out a luminous reaction with the use of the resulting ATP and one or more enzymes; and

analyzing the amounts of an amplified product based on the quantity of light emitted as a result of the luminous reaction to thereby type the single-nucleotide polymorphism.

13. The method according to claim 10, wherein the oligonucleotide probe has at least one non-complementary sequence being not complementary with any of possible sequences of one or more mutation sites in a region of the target nucleic acid corresponding to the probe other than the single-nucleotide polymorphism.

14. A kit for amplifying a nucleic acid and/or for analyzing a single-nucleotide polymorphism, comprising an oligonucleotide primer or probe being capable of complementarily hybridizing with a specific region of a target nucleic acid containing at least one mutation site,

wherein the oligonucleotide primer or probe has at least one non-complementary sequence being not complementary to any of possible sequences of the at least one mutation site.