Method for designing probe in dna microarray, and dna microarray provided with probe designed thereby

Abstract

Provided is a probe to be used in a DNA microarray having an excellent detection rate of a polymorphism such as SNP contained in genomic DNA. A method for designing a probe according to the invention includes the steps of: specifying one or more regions covering at least a part of fragments flanked by restriction enzyme recognition sites recognized by a restriction enzyme, contained in genomic DNA derived from an organism to be tested; and designing a probe for the specified one or more regions for detecting the fragment in the organism to be tested.

1 - 20 . (canceled) 
     
     
         21 . A method for designing a probe, comprising the steps of:
 specifying one or more regions having a shorter nucleotide length than genomic DNA fragment flanked by restriction enzyme recognition sites, contained in genomic DNA derived from a target organism, and covering at least one portion of the genomic DNA fragment; and   designing the specified one or more regions as a probe for detecting a mutation contained in the genomic DNA fragment.   
     
     
         22 . The method for designing a probe according to  claim 21 , wherein the one or more regions is specified by performing the following steps:
 ( 1   a ) extracting the genomic DNA;   ( 1   b ) digesting the extracted genomic DNA with the restriction enzyme;   ( 1   c ) connecting an adaptor to the genomic DNA fragments obtained the step ( 1   b );   ( 1   d ) amplifying the genomic DNA fragments using a primer capable of hybridizing to the adaptor;   ( 1   e ) sequencing the amplified genomic DNA fragments; and   ( 1   f ) determining the one or more regions based on the nucleotide sequence.   
     
     
         23 . The method for designing a probe according to  claim 22 , wherein, in the step ( 1   b ), the genomic DNA is digested with more than one restriction enzyme. 
     
     
         24 . The method for designing a probe according to  claim 23 , wherein, in the step ( 1   c ), an adaptor is connected corresponding to one restriction enzyme selected from the more than one restriction enzyme or corresponding to a part of the more than one restriction enzyme used. 
     
     
         25 . The method for designing a probe according to  claim 22 , wherein the adaptor has a complementary sequence to a protruding end of the genomic DNA fragments obtained in the step ( 1   b ). 
     
     
         26 . The method for designing a probe according to  claim 21 , wherein the one or more regions are specified using the nucleotide sequence data on the genomic DNA by performing the following steps:
 ( 2   a ) searching the nucleotide sequence data on the genomic DNA for the restriction enzyme recognition sequence to specify the nucleotide sequence of the genomic DNA fragments obtained by digesting the genomic DNA with the restriction enzyme; and   ( 2   b ) determining the one or more regions based on the specified nucleotide sequence.   
     
     
         27 . The method for designing a probe according to  claim 26 , wherein, in the step ( 2   a ), the genomic DNA fragments obtained by digesting the genomic DNA with more than one restriction enzyme are sequenced. 
     
     
         28 . The method for designing a probe according to  claim 27 , wherein, in the step ( 2   b ), the one or more regions are determined with respect to the genomic DNA fragments flanked by one restriction enzyme selected from the more than one restriction enzyme or a part of more than one restriction enzyme used. 
     
     
         29 . The method for designing a probe according to  claim 21 , wherein the one or more regions are determined by performing the following steps:
 ( 3   a ) extracting the genomic DNA;   ( 3   b ) digesting the extracted genomic DNA with the restriction enzyme;   ( 3   c ) connecting an adaptor to the genomic DNA fragments obtained in the step ( 3   b );   ( 3   d ) amplifying the genomic DNA fragments using a primer capable of hybridizing to the adaptor;   ( 3   e ) digesting the amplified genomic DNA fragment with another restriction enzyme; and   ( 3   f ) separating the DNA fragments obtained by digestion in the step ( 3   e ) as probes.   
     
     
         30 . The method for designing a probe according to  claim 29 , wherein, in the step ( 3   b ), the genomic DNA is digested with more than one restriction enzyme. 
     
     
         31 . The method for designing a probe according to  claim 30 , wherein, in the step ( 3   c ), an adaptor is connected corresponding to one restriction enzyme selected from the more than one restriction enzyme or corresponding to a part of the more than one restriction enzyme used. 
     
     
         32 . The method for designing a probe according to  claim 29 , wherein, the adaptor has a complementary sequence to a protruding end of the genomic DNA fragments obtained in the step ( 3   b ). 
     
     
         33 . The method for designing a probe according to  claim 21 , wherein the designed probe has a 20 to 100 nucleotide length. 
     
     
         34 . A DNA microarray comprising a probe designed by the method for designing a probe according to  claim 21  and a carrier on which the probe to be immobilized. 
     
     
         35 . The DNA microarray according to  claim 34 , wherein the probe is synthesized on the carrier based on the sequence data. 
     
     
         36 . A method for detecting a mutation using a DNA microarray, comprising the steps of:
 extracting a genomic DNA derived from an organism to be tested;   digesting the genomic DNA with a restriction enzyme having the same recognition sequence as the restriction enzyme used in the designing a probe immobilized on the DNA microarray according to  claim 34 ;   connecting an adaptor to the genomic DNA fragments obtained by the restriction enzyme treatment;   amplifying the genomic DNA fragments using a primer capable of hybridizing to the adaptor; and   detecting a hybrid of the genomic DNA fragment with the probe by bringing the amplified genomic DNA fragment into contact with the DNA microarray according to  claim 34 .   
     
     
         37 . The method for detecting a mutation using the DNA microarray according to  claim 36 , wherein, in the step of digesting the genomic DNA, the genomic DNA is digested with more than one restriction enzyme. 
     
     
         38 . The method for detecting a mutation using a DNA microarray according to  claim 37 , wherein, in the step of connecting an adaptor is connected corresponding to one restriction enzyme selected from the more than one restriction enzyme or corresponding to a part of the more than one restriction enzyme are connected. 
     
     
         39 . The method for detecting a mutation using a DNA microarray according to  claim 36 , wherein the adaptor has a complementary sequence to a protruding end of the genomic DNA fragments obtained in the step of digesting the genomic DNA with a restriction enzyme. 
     
     
         40 . The method for detecting a mutation using a DNA microarray according to  claim 36 , wherein the organism to be tested is different from the organism used in preparing the DNA microarray.