HLA-DQB1 typing using DNA microarray.

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I tried to design an oligonucleotide microarray to identify HLA-DQB1 alleles based on the HLA sequence data base in 2002 year. The result was obtained described as below.1.MaterialsDNA was extracted from blood of volunteers using phenol/chloroform method. HLA-DQB1 alleles were previously typed by PCR-SSP method and direct sequence method.2.PrimersThe primers to amplify HLA-DQB1 alleles in Exon2 and Exon3 were designed. One sense primer and two antisense primers were designed for Exon2 and one primer pair was designed for Exon3. These primers were labeled with Biotin at the site of 5' end and mixed for multiplex PCR. Two PCR products were detected using the primer mixture to amplify Exon2 and Exon3 by the agarose gel electrophoresis. The primer mixture was used for the DNA microarray analysis.3.Capture DNA (oligonucleotide probe)Sequence-specific 24 captures for Exon2 and 2 captures for Exon3 to identify the alleles were designed. DNA microarray was prepared by spotting these captures (oligonucleotide probes) on the glass. HIA-DQB1 typing was tried by this DNA microarray.4.HLA-DQB1 typing from blood using DNA microarray.HLA-DQB1 typing was tried using DNA microarray immobilized 26 oligonucleotide probes. The method was such as below. The target DNA was amplified with primer mixture labeled with biotin to amplify Exon2 and Exon3 of HLA-DQB1 alleles. PCR products were denatured and hybridized with capture DNAs. After conjugating with streptavidin-biotin labeled peroxidase solution, color development was performed with peroxidase substrate kit TMB. The allele type was determined by the pattern of color development. The allele specific pattern was detected, but some false positive reaction was also obtained. The probes to show these false positive reactions should be redesigned.