Method of amplifying nucleic acids, reagent kit for amplifying nucleic acids, method of detecting single nucleotide polymorphism, and reagent kit for detecting single nucleotide polymorphism

Abstract

An object of the present invention is to provide a nucleic acid amplification method for amplifying a desired nucleic acid while suppressing amplification of byproducts in a PCR reaction, a reagent kit used for nucleic acid amplification, a method of detecting single nucleotide polymorphism to detect single nucleotide polymorphism by utilizing that amplification of byproducts is suppressed in a PCR reaction, and a reagent kit used for detecting single nucleotide polymorphism. The method of amplifying nucleic acids by PCR is characterized by admixing in a reaction solution, a homologous recombinant protein which contains at least one of a RecA protein derived from Thermus thermophilus , and a modified RecA protein obtained by modification of the RecA protein and having a function similar to that of the RecA protein, and carrying out PCR.

1 . A method of amplifying nucleic acids by PCR, characterized by admixing in a reaction solution, a homologous recombinant protein which contains at least one of a RecA protein derived from  Thermus thermophilus , and a modified RecA protein obtained by modification of the RecA protein and having a function similar to that of the RecA protein, and carrying out PCR.  
     
     
         2 . The method of amplifying nucleic acids according to  claim 1 , characterized by adding ATP-γS to the reaction solution and carrying out PCR.  
     
     
         3 . The method of amplifying nucleic acids according to  claim 1 , characterized by the use of a template DNA having a region of inhibitory or suppressive secondary structure.  
     
     
         4 . The method of amplifying nucleic acids according to  claim 1 , characterized by adding KCl to the reaction solution and carrying out PCR.  
     
     
         5 . The method of amplifying nucleic acids according to  claim 1 , characterized by adding Mg 2+  to the reaction solution and carrying out PCR.  
     
     
         6 . The method of amplifying nucleic acids according to  claim 1 , characterized by adding a plurality of sets of primer DNAs to the reaction solution and carrying out PCR.  
     
     
         7 . A method of amplifying nucleic acids by PCR, characterized by admixing in a reaction solution, a homologous recombinant protein which contains at least one of a RecA protein which causes primer extension reaction only for a primer DNA having a mismatch of 3 bases or less with a template DNA, and a modified RecA protein obtained by modification of the RecA protein and having a function similar to that of the RecA protein, and carrying out PCR.  
     
     
         8 . The method of amplifying nucleic acids according to  claim 7 , characterized by adding ATP-γS to the reaction solution and carrying out PCR.  
     
     
         9 . The method of amplifying nucleic acids according to  claim 7 , characterized by the use of a template DNA having a region of inhibitory or suppressive secondary structure.  
     
     
         10 . The method of amplifying nucleic acids according to  claim 7 , characterized by adding KCl to the reaction solution and carrying out PCR.  
     
     
         11 . The method of amplifying nucleic acids according to  claim 7 , characterized by adding Mg 2+  to the reaction solution and carrying out PCR.  
     
     
         12 . The method of amplifying nucleic acids according to  claim 7 , characterized by adding a plurality of sets of primer DNAs to the reaction solution and carrying out PCR.  
     
     
         13 . A reagent kit for amplifying nucleic acids by PCR, characterized by comprising 
 a DNA polymerase,    four kinds of dNTPs,    a buffer solution, and    a homologous recombinant protein which contains at least one of a RecA protein derived from  Thermus thermophilus  and a modified RecA protein obtained by modification of the RecA protein and having a function similar to that of the RecA protein.    
     
     
         14 . The reagent kit for amplifying nucleic acids according to  claim 13 , characterized by comprising ATP-γS.  
     
     
         15 . The reagent kit for amplifying nucleic acids according to  claim 13 , characterized by comprising KCl.  
     
     
         16 . The reagent kit for amplifying nucleic acids according to  claim 13 , characterized by comprising Mg 2+ .  
     
     
         17 . A method of detecting single nucleotide polymorphism, wherein the method is characterized by admixing in a reaction solution, a homologous recombinant protein which contains at least one of a RecA protein derived from  Thermus thermophilus , and a modified RecA protein obtained by modification of the RecA protein and having a function similar to that of the RecA protein, and carrying out PCR, by using a primer DNA corresponding to a sequence containing a base which forms single nucleotide polymorphism in a template DNA, to detect single nucleotide polymorphism by amplifying a desired nucleic acid.  
     
     
         18 . The method of detecting single nucleotide polymorphism according to  claim 17 , characterized by adding ATP-γS to the reaction solution and carrying out PCR.  
     
     
         19 . The method of detecting single nucleotide polymorphism according to  claim 17 , characterized by adding KCl to the reaction solution and carrying out PCR.  
     
     
         20 . The method of detecting single nucleotide polymorphism according to  claim 17 , characterized by adding Mg 2+  to the reaction solution and carrying out PCR.  
     
     
         21 . A reagent kit for detecting single nucleotide polymorphism, characterized by comprising 
 a DNA polymerase,    four kinds of dNTPs,    a buffer solution, and    a homologous recombinant proteins which contains at least one of a RecA protein derived from  Thermus thermophilus  and a modified RecA protein obtained by modification of the RecA protein and having a function similar to that of the RecA protein.    
     
     
         22 . The reagent kit for detecting single nucleotide polymorphism according to  claim 21 , characterized by comprising ATP-γS.  
     
     
         23 . The reagent kit for detecting single nucleotide polymorphism according to  claim 21 , characterized by comprising KCl.  
     
     
         24 . The reagent kit for detecting single nucleotide polymorphism according to  claim 21 , characterized by comprising Mg 2+ .