Method for detecting mutant dna

Abstract

The present invention relates to a method for detecting of a mutant DNA using a probe, comprising: (1) contacting a sample containing a single-stranded DNA which has a substituted nucleotide, a deleted nucleotide region, or an inserted nucleotide region (mutant-type DNA), or/and a wild-type single-stranded DNA (wild-type DNA) corresponding thereto with the probe which hybridizes with both single-stranded DNA, to form a hybrid with the mutant-type DNA (mutant-type hybrid) or/and a hybrid with a wild-type DNA (wild-type hybrid), wherein at least one of the obtained mutant-type hybrid and wild-type hybrid has the stem structure; (2) separating the obtained mutant-type hybrid or/and wild-type hybrid by electrophoresis on the basis of presence or absence of the stem structure or difference in the stem structure; and (3) detecting the presence or absence of the mutant-type DNA in the sample.

1 - 22 . (canceled) 
     
     
         23 . A method for detecting whether a wild-type nucleotide region is deleted by a deletion mutation at a suspected mutated nucleotide region in a target DNA sequence in a sample, the method comprising:
 (1) providing a probe having a structure comprising   3′ 1stSS 211 -X′ 211 -DM′-X′ 212 -2ndSS 211  5′;   
       wherein:
 the length of the probe is 30 to 300 nucleotides; 
 1stSS 211  is a nucleotide sequence complimentary to a portion of the target DNA sequence located on the 5′ side of the suspected deletion mutation region and 2ndSS 211  is a nucleotide sequence complimentary to a portion of the target DNA sequence located on the 3′ side of the suspected deletion mutation region; 
 each of X′ 211  and X′ 212  independently represents a deoxyribonucleotide A, T, C or G; and 
 DM′ is a nucleotide sequence 4 to 200 nucleotides in length that hybridizes with the region of the target DNA sequence that is present in the target DNA with a wild-type sequence and deleted from the target DNA with the deletion mutation, DM′ has the same length as the length of the suspected deletion mutation region, and DM′ contains a nucleotide sequence of 4 to 60 nucleotides that is capable of forming a double-stranded stem structure of 2 to 20 base pairs when the probe is hybridized with the target DNA sequence; 
 (2) contacting the probe with the sample containing the target DNA sequence in a solution under conditions capable of forming probe/target DNA sequence hybrids, wherein there is an electrophoretic mobility difference between the probe/target DNA sequence hybrids when the target DNA sequence has the wild-type sequence and the probe/target DNA sequence hybrids when the target DNA sequence has the deletion mutation; 
 (3) analyzing the probe/target DNA sequence hybrids formed in step (2) by an electrophoresis which is performed under conditions in which there is an electrophoretic mobility difference between the probe/target DNA sequence hybrids when the target DNA sequence has the wild-type sequence and the probe/target DNA sequence hybrids when the target DNA sequence has the deletion mutation, thereby obtaining an electrophoresis analysis result; and 
 (4) determining whether the wild-type nucleotide region is deleted by the deletion mutation at the suspected mutated nucleotide region in the target DNA sequence in the sample by comparing the electrophoresis analysis result of step (3) with a first electrophoresis analysis result when the target DNA sequence has the wild-type sequence or/and a second electrophoresis analysis result when the target DNA sequence has the deletion mutation, wherein the first electrophoresis analysis result or/and the second electrophoresis analysis result is/are obtained by performing steps (1), (2) and (3). 
 
     
     
         24 . The method according to  claim 23 , wherein the electrophoresis is microchip electrophoresis or capillary gel electrophoresis. 
     
     
         25 . A method for detecting whether a wild-type nucleotide region is deleted by a deletion mutation at a suspected mutated nucleotide region in a target DNA sequence in a sample, the method comprising: 
       (1) providing a probe having a structure comprising
 3′ 1stSS 221 -X′ 221 -DM′-Stp 221 -X′ 222 -2ndSS 221  5′; 
 
       wherein:
 the length of the probe is 30 to 300 nucleotides; 
 1stSS 221  is a nucleotide sequence complimentary to a portion of the target DNA sequence located on the 5′ side of the suspected deletion mutation region and 2ndSS 221  is a nucleotide sequence complimentary to a portion of the target DNA sequence located on the 3′ side of the suspected deletion mutation region; 
 each of X′ 221  and X′ 222  independently represents a deoxyribonucleotide A, T, C or G; 
 DM′ is a nucleotide sequence 1 to 200 nucleotides in length that hybridizes with the region of the target DNA sequence that is present in the target DNA with a wild-type sequence and deleted from the target DNA with the deletion mutation, DM′ has the same length as the length of the suspected deletion mutation region, and DM′-Stp 221  contains a nucleotide sequence of 4 to 60 nucleotides that is capable of forming a double-stranded stem structure of 2 to 20 base pairs when the probe is hybridized with the target DNA sequence; 
 (2) contacting the probe with the sample containing the target DNA sequence in a solution under conditions capable of forming probe/target DNA sequence hybrids, wherein there is an electrophoretic mobility difference between the probe/target DNA sequence hybrids when the target DNA sequence has the wild-type sequence and the probe/target DNA sequence hybrids when the target DNA sequence has the deletion mutation; 
 (3) analyzing the probe/target DNA sequence hybrids formed in step (2) by an electrophoresis which is performed under conditions in which there is an electrophoretic mobility difference between the probe/target DNA sequence hybrids when the target DNA sequence has the wild-type sequence and the probe/target DNA sequence hybrids when the target DNA sequence has the deletion mutation, thereby obtaining an electrophoresis analysis result; and 
 (4) determining whether the wild-type nucleotide region is deleted by the deletion mutation at the suspected mutated nucleotide region in the target DNA sequence in the sample by comparing the electrophoresis analysis result of step (3) with a first electrophoresis analysis result when the target DNA sequence has the wild-type sequence or/and a second electrophoresis analysis result when the target DNA sequence has the deletion mutation, wherein the first electrophoresis analysis result or/and the second electrophoresis analysis result is/are obtained by performing steps (1), (2) and (3). 
 
     
     
         26 . The method according to  claim 25 , wherein the electrophoresis is microchip electrophoresis or capillary gel electrophoresis.