Method of detecting gene mutation

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.

1 . A method of detecting a base sequence, comprising the steps of: amplifying DNA containing a target base sequence to be detected having a mutation site using DNA polymerase; hybridizing the amplified DNA to a hybridization probe having a base sequence complementary to the target base sequence to be detected; and detecting a hybrid formed by the hybridization, 
 wherein at least one of 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, the 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 the hybrid is detected by affinity chromatography with the use of the first and second labeling agents.    
     
     
         2 . The method according to  claim 1 , wherein the mutation site is a point mutation, and the reaction solution for effecting the DNA amplification further contains an unlabeled oligonucleotide having a base sequence different in a single base at the position of the point mutation from the base sequence of the labeled hybridization probe, in an amount sufficient to enhance the specificity of hybridization of the amplified DNA to the hybridization probe.  
     
     
         3 . The method according to  claim 1  or  2 , wherein the DNA amplification is carried out by PCR.  
     
     
         4 . A kit comprising: primers for amplifying DNA containing a target base sequence to be detected having a mutation site using DNA polymerase; a hybridization probe having a base sequence complementary to the target base sequence to be detected; and a test strip for 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, the hybridization probe is labeled with a second labeling agent, the base sequence of the hybridization probe is designed not to inhibit the DNA amplification, and the test strip allows of detection of a hybrid of the amplified DNA and the hybridization probe with the use of the first and second labeling agents.    
     
     
         5 . The kit according to  claim 4 , wherein the mutation site is a point mutation and the kit further comprises an unlabeled oligonucleotide having a base sequence different in a single base at the position of the point mutation from the base sequence of the labeled hybridization probe.  
     
     
         6 . The kit according to  claim 4  or  5 , wherein the primers are primers for PCR.