SNP detection method and SNP detection apparatus

• US 20030049628A1
• Filed: 09/05/2001
• Published: 03/13/2003
• Est. Priority Date: 09/28/2000
• Status: Active Grant

• Current Assignee: Hitachi, Ltd.
• Original Assignee: Hitachi, Ltd.
• Inventors: Kamahori, Masao, Kambara, Hideki, Okano, Kazunori, Zhou, Guohua
• Granted Patent: US 6,750,018 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer

Abstract

To provide a method and an apparatus for detecting DNA mutation without using electrophoresis, the presence or absence of specific sequence is detected by a method comprising a step of serially hybridizing a 5-base-long to 8-base-long short oligomer 6, which is capable of being engaged in the complementary strand extension reaction, and a long oligomer 61, which is capable of hybridizing with a target DNA but incapable of being engaged in the complementary strand extension reaction, with the target DNA 63; a step of carrying out the complementary strand extension reaction 66 starting from the short primer using four kinds of nucleic acid substrates and polymerase; and a step of detecting photo-emission, in which pyrophosphate 68 produced as a reaction by-product of the complementary strand extension reaction is converted into ATP and the photo-emission reaction is carried out using an enzyme.

Claims

1. A DNA mutation detection method characterized in that a primer which has a sequence complementary to a region of a specific sequence containing anticipated mutations in a target DNA hybridizes with a sample and whether a complementary strand extension reaction with nucleic acid substrates and polymerase starting from said primer proceeds or not is detected by detecting chemiluminescence utilizing pyrophosphate produced as a reaction by-product of the complementary strand extension to detect the presence or absence of said specific sequence.

2. A DNA mutation detection method as claimed in claim 1 wherein multiple kinds of said nucleic acid substrates or analogues thereof are simultaneously added and said complementary strand extension reaction is repeated.

3. A DNA mutation detection method as claimed in claim 1 wherein four kinds of said nucleic acid substrates, which are capable of being engaged in the complementary strand extension, or analogues thereof are simultaneously added.

4. A DNA mutation detection method as claimed in claim 1 wherein the complementary strand extension is proceeded or not depending on whether the end base of said primer mismatches or matches with the base of the site of mutation and further the primer is designed to have a nucleotide which is not complementary to the corresponding site of said target DNA in the vicinity of its 3′

-end so as to more securely control the complementary strand extension in response to the match or mismatch of the 3′

end of the primer to detect single nucleotide polymorphisms (SNPs).

5. A DNA mutation detection method as claimed in claim 1 wherein the extended complementary strand by said complementary strand extension reaction is decomposed with an enzyme and said primer is repeatedly hybridized with said target DNA to proceed said complementary strand extension reaction and pyrophosphate produced in response to the extension reaction is detected.

6. A DNA mutation detection method as claimed in claim 5 wherein the enzyme to decompose said extended complementary strand is an enzyme which decompose a DNA strand from the 5′

-end.

8. A reagent kit used for a method of claim 1 comprising at least a DNA primer, which is complementary to said target DNA and the 3′

-end of which is designed to be hybridizable at a site of single nucleotide polymorphism of interest or the vicinity thereof, and nucleic acid substrates.

9. A reagent kit used for a method of claim 1 characterized in that said kit comprises a DNA primer, which is complementary to said target DNA and the 3′

-end of which is designed to be hybridizable at a site of single nucleotide polymorphism of interest or the vicinity thereof;

nucleic acid substrates; and

an enzyme to decompose pyrophosphate contained as an impurity, and that said kit is used for detecting single polymorphisms (SNPs).

7. A single nucleotide polymorphism detection method characterized in that a DNA primer, which is complementary to a target DNA and is hybridizable at the 3′

-end with a site of single nucleotide polymorphism of interest or the vicinity thereof, is used to proceed a complementary strand extension reaction starting from said primer, which is designed to successively incorporate at least multiple nucleic acid substrates, chemiluminescence is generated utilizing pyrophosphate produced as a result of the reaction, and the photo-emission is detected thereby detecting single nucleotide polymorphisms (SNPs).

10. A method for detecting single nucleotide polymorphisms characterized in that two kinds of primers having the end nucleotide respectively complementary to wild type and mutant DNA samples are held separately in different cells on the solid surface or different reaction chambers by kind of said primers, and a mixture of multiple kinds of dNTP is added to said cells or a reaction solution of said reaction chambers to provide conditions where a complementary strand extension reaction starting from said primers can take place, pyrophosphate produced as a result of actual complementary strand extension reaction starting from said primers which hybridize with a target DNA is converted into ATP, and chemiluminescence produced by reacting the ATP with luciferin or the like is detected to discriminate the kind of primers which are engaged in the complementary strand extension reaction, whereby single nucleotide polymorphisms in said target DNA are detected.

11. A method for detecting single nucleotide polymorphisms characterized in that multiple kinds of long oligomers, which are different in sequence from each other and are capable of hybridizing with a target DNA but incapable of proceeding a complementary strand extension reaction, are held separately by kind of said oligomers in different cells on the solid surface or in different reaction chambers, 5-base-long to 8-base-long short oligomers capable of proceeding the complementary strand extension reaction are added to said cells or said reaction chambers, said short oligomers and said long oligomers are serially hybridized with said target DNA, the complementary strand extension reaction starting from the short primers proceeds by further adding a mixture of multiple kinds of dNTP, pyrophosphate produced as a result of the reactions is converted into ATP, and chemiluminescence produced by reacting the ATP with a chemiluminescence reagent is detected to discriminate the kind of the primers which are engaged in the complementary strand extension reaction, whereby single nucleotide polymorphisms (SNPs) contained in said target DNA or the ratio of DNA substitutions in samples can be detected.

12. A mutation detection apparatus for detecting DNA mutations characterized in that said apparatus comprises a reaction chip furnished with multiple cells on the solid surface, which hold at least two different primers having end nucleotides each complementary to wild type and mutant DNA samples, separately by kind of said primers, or a reaction chamber supporting base on which multiple reaction chambers holding said primers separately by kind of said primers are placed;

and a light detector to detect chemiluminescence which is generated by reacting ATP with a chemiluminescence reagent, said ATP being converted from pyrophosphate produced as a result of actual complementary strand extension reaction starting from said primers hybridized with said target DNA under the conditions where the complementary strand extension reaction starting from the primers can take place by adding a mixture of multiple kinds of dNTP to said cells or a reaction solution in said reaction chambers, whereby the kind of said primers which are engaged in said complementary strand extension reaction is discriminated to detect mutations contained in said target DNA.

13. A mutation detection apparatus for detecting DNA mutations characterized in that said apparatus comprises a reaction chip furnished with multiple cells on the solid surface, which hold one or more kinds of long oligomers, which are capable of hybridizing with a target DNA but incapable of being engaged in a complementary strand extension reaction and are different in sequence from each other, separately by kind of said oligomers, or a reaction chamber supporting base on which multiple reaction chambers holding said long oligomers separately by kind of said oligomers are placed;

and a light detector to detect chemiluminescence which is generated by reacting ATP with a chemiluminescence reagent, said ATP being converted from pyrophosphate produced by a serial hybridization of short oligomers and said long oligomers with the target DNA by adding 5-base-long to 8-base-long short oligomers capable of being engaged in the complementary strand extension reaction respectively to said cells or said reaction chambers followed by the subsequent complementary strand extension reaction starting from said short primers by further adding a mixture of multiple kinds of dNTP, whereby the kind of said short primers which are engaged in said complementary strand extension reaction is discriminated to detect mutations contained in said target DNA.

14. A method for detecting DNA mutations comprising (1) a step of hybridizing a primer which contains a sequence complementary to a region of a specific sequence of a target DNA;

(2) a step of carrying out a complementary strand extension reaction starting from said primer using four kinds of nucleic acid substrates and polymerase; and

(3) a step of detecting photo-emission in which pyrophosphate produced as a reaction by-product of said complementary strand extension reaction is converted into ATP and a photo-emission reaction is carried out using an enzyme, whereby the presence or absence of said specific sequence is detected.

15. A method for detecting mutations comprising (1) a step of hybridizing a primer which contains a sequence complementary to a region of a specific sequence of a target DNA and a sequence not complementary to the sequence of the target DNA at the second base or the third base from the 3′

-end;

(2) a step of carrying out a complementary strand extension reaction starting from said primer using four kinds of nucleic acid substrates and polymerase; and

(3) a step of detecting photo-emission in which pyrophosphate produced as a reaction by-product of said complementary strand extension reaction is converted into ATP and a photo-emission reaction is carried out using an enzyme, whereby the presence or absence of said specific sequence is detected.

16. A method for detecting mutations comprising (1) a step of serially hybridizing a 5-base-long to 8-base-long short oligomer, which are capable of being engaged in a complementary strand extension reaction, and a long oligomer, which is capable of hybridizing with a target DNA but incapable of being engaged in a complementary strand extension reaction, with the target DNA;

(2) a step of carrying out the complementary strand extension reaction starting from said short primer using four kinds of nucleic acid substrates and polymerase; and

(3) a step of detecting photo-emission in which pyrophosphate produced as a reaction by-product of said complementary strand extension reaction is converted into ATP and a photo-emission reaction is carried out using an enzyme, whereby the presence or absence of said specific sequence is detected.

17. A method characterized in that a sample is examined using at least two probes or primers which contain a part of a sequence of a DNA region of interest and correspond to a wild type and a mutant respectively, and the presence or absence of mutations or the ratio of mutations are examined by comparing results thus obtained.

18. A method as claimed in claim 17 wherein the wild type and the mutant are discriminated at least by the difference in kind of bases at the end of the probes or the primers.

19. A method as claimed in claim 17 wherein the wild type and the mutant are discriminated by a 5 mar or a 6 mar which is respectively complementary and contains a site of mutation.

20. A method for detecting SNPs as claimed in claim 17 wherein the primers or the probes for discriminating mutants are hybridized with the target DNA, the complementary strands are synthesized, and the resulting pyrophosphate is detected.

21. A method for detecting SNPs as claimed in claim 17 wherein the primers or the probes for discriminating mutants are hybridized with the target DNA, the complementary strands are synthesized, and the resulting synthesized DNA strands are analyzed.