Method for typing of HLA class I alleles

Abstract

This invention provides a method, a kit and a reagent for typing of the HLA class I alleles. Explaining concretely, a single HLA class I antigen or allele is determined by combining PCR amplification using a primer pair which can amplify all HLA-A alleles, all HLA-B alleles or all HLA-C alleles, or which is specific to the common sequence to alleles of the specific group consisting of the specific HLA-A alleles or the specific HLA-B alleles, with reverse hybridization analysis using DNA probes capable of specifically hybridizing with the sequence of al least a specific HLA-A allele, at least a specific HLA-B allele or at least a specific HLA-C allele, which are covalently immobilized on wells of microtiter plates.

1 . A method for detecting a specific base sequence, wherein hybridization is performed in a hybridization buffer containing 10% to 25% formamide, at 32° C. to 42° C., using a probe of 14 to 24 or more of bases.  
     
     
         2 . The method claimed in  claim 1 , wherein the hybridization buffer contains 0.25M di-sodium hydrogenphosphate, 7% sodium dodecyl sulfate, 1% bovine serum albumin, 0.03M phosphoric acid, 0.5M ethylenediaminetetraacetic acid and 10% to 25% formamide.  
     
     
         3 . The method claimed in  claim 1  or  2 , wherein the temperature for washing after the hybridization is room temperature.  
     
     
         4 . The method claimed in  claim 1 , wherein the probes are hybridized with amplified products by the PCR method.  
     
     
         5 . The method claimed in  claim 4 , wherein at least one of the primer pair is labeled.  
     
     
         6 . The method claimed in  claim 1 , wherein nucleic acids are hybridized with the probes immobilized on a support.  
     
     
         7 . The method claimed in  claim 4 , which comprises hybridizing the amplified products obtained by the PCR method with the immobilized DNA probes, adding an enzyme-conjugate which specifically bonds to a label of the amplified products thereto at the same time or after the hybridization, and adding a chromogenic substrate, a luminescent substrate or a fluorescent substrate to the mixture, to detect as signals whether or not the amplified products are hybridized with the immobilized DNA probes.  
     
     
         8 . The method claimed in  claim 7 , wherein the label is a biotin and the enzyme-conjugate is an enzyme-conjugated streptavidin.