Method of detecting gene mutation

• US 9,677,127 B2
• Filed: 05/12/2008
• Issued: 06/13/2017
• Est. Priority Date: 11/07/2002
• Status: Active Grant

• Current Assignee: Sekisui Medical Company Limited (Sekisui), Shigeo Kure, Sigeo Kure, Yoichi Matsubara
• Original Assignee: Sekisui Medical Company Limited (Sekisui)
• Inventors: Matsubara, Yoichi, Kure, Shigeo
• Primary Examiner(s): SALMON, KATHERINE D
• Application Number:
  US12/119,141
• Publication Number:
  US 20080318238A1
• Time in Patent Office: 3,319 Days
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 2535/131 Allele specific probes

Abstract

DNA amplification and hybridization are successively carried out in a reaction system containing primers for the DNA amplification and hybridization probes, followed by detecting the hybrid in the reaction solution by affinity chromatography, wherein at least one of the primers to be used in the DNA amplification is labeled with a first labeling agent so that the amplified DNA will be labeled with the first labeling agent, a hybridization probe is labeled with a second labeling agent and contained in a reaction solution for effecting the DNA amplification, the base sequence of the hybridization probe is designed not to inhibit the DNA amplification, and a hybrid is detected by affinity chromatography with the use of the first and second labeling agents.

Claims

1. A method of detecting a first sequence or a second sequence in a target region of DNA, comprising:

(A) providing a double stranded DNA comprising a target region, the target region comprising either a first sequence or a second sequence differing at a mutation site;

(B) amplifying the double stranded DNA in a first reaction mixture by a DNA polymerase reaction comprising

(i) a first DNA polymerase,

(ii) a first primer complementary to a second strand of the double stranded DNA and labeled with a first labeling agent,

(iii) a second primer complementary to a first strand of the double stranded DNA,

(iv) a first hybridization probe labeled with a second labeling agent and designed not to inhibit the DNA amplification, the first hybridization probe comprising

a length of 10-15 mer,

the first sequence mutation site, and

full complementarity to the first strand of the double stranded DNA comprising the first sequence and

(v) a second hybridization probe not labeled with the second labeling agent and designed not to inhibit the DNA amplification, the second hybridization probe comprising

a length of 10-15 mer;

the second sequence mutation site, and

full complementarity to the first strand of the double stranded DNA comprising the second sequence,

thereby obtaining a first amplified DNA comprising the first labeling agent; and

(C) hybridizing at 25°

C. the first amplified DNA in the first reaction mixture to the first hybridization probe or the second hybridization probe, wherein hybridizing occurs in the first reaction mixture and no addition of a reagent occurs between amplifying and hybridizing;

(D) applying the first reaction mixture after hybridization onto a first chromatography support; and

(E) amplifying the double stranded DNA in a second reaction mixture by a DNA polymerase reaction comprising

(i) a second DNA polymerase,

(ii) the first primer labeled with the first labeling agent,

(iii) the second primer,

(iv) the first hybridization probe not labeled with the second labeling agent, and

(v) the second hybridization probe labeled with the second labeling agent,

thereby, obtaining a second amplified DNA comprising the first labeling agent; and

(F) hybridizing at 25°

C. the second amplified DNA in the second reaction mixture to the first hybridization probe or the second hybridization probe, wherein hybridizing occurs in the second reaction mixture and no addition of a reagent occurs between amplifying and hybridizing;

(G) applying the second reaction mixture after hybridization onto a second chromatography support; and

(H) determining that the DNA comprises the first sequence by detecting a first hybrid via affinity chromatography on the first chromatography support, the first hybrid being fully complementary between (i) the first hybridization probe labeled with the second labeling agent and (ii) DNA comprising the first sequence labeled with the first labeling agent;

or

(I) determining that the DNA comprises the second sequence by detecting a second hybrid via affinity chromatography on the second chromatography support, the second hybrid being fully complementary between (i) the second hybridization probe labeled with the second labeling agent and (ii) DNA comprising the second sequence labeled with the first labeling agent,

wherein, a ligase enzyme is not required after hybridization.

2. The method according to claim 1, wherein a mutation of the mutation site is a point mutation, and the second hybridization probe in the first reaction mixture and the first hybridization probe in the second reaction mixture are comprised in an amount sufficient to enhance specificity of hybridization of the first amplified DNA and the second amplified DNA to the first hybridization probe and the second hybridization probe.

3. The method according to claim 1, wherein the DNA amplifications are carried out by PCR.

4. The method according to claim 1 wherein the first hybridization probe and the second hybridization probe are 10-13 mers.

5. The method according to claim 4 wherein the first hybridization probe and the second hybridization probe are 11-13 mers.

6. The method according to claim 5 wherein the first hybridization probe is a 12 mer or a 13 mer and the second hybridization probe is a 12 mer or a 13 mer.

7. The method according to claim 1 wherein the length of the first hybridization probe and the second hybridization probe are selected so that hybridization to the first amplified DNA and the second amplified DNA, respectively, does not occur at a temperature at which the first DNA polymerase and the second DNA polymerase are actively amplifying the double stranded DNA.

8. The method according to claim 1 wherein a mutation of the mutation site is an insertion mutation.

9. The method according to claim 1 wherein a mutation of the mutation site is a deletion mutation.

10. The method according to claim 1 wherein the second hybridization probe in the first reaction mixture contains a sequence complimentary to the mutation site of the target region at a middle of the second hybridization probe and the first hybridization probe in the second reaction mixture contains a sequence complimentary to the mutation site of the target region at a middle of the first hybridization probe.

11. The method according to claim 1 wherein the first hybridization probe and the second hybridization probe are 10 mers.

12. The method according to claim 1 wherein the first hybridization probe and the second hybridization probe are 11 mers.

13. The method of claim 1, wherein no addition of a reagent occurs between hybridizing and applying the first reaction mixture on to the first chromatography support and applying the second reaction mixture on to the second chromatography support.