Development of automated high-resolutoin HLA-DNA typing system using DNA tips

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In order to establish the automated high-resolution HLA typing system, we have developed the PCR-SSOP (polymerase chain reaction - sequence specific oligonucleotide probe) method using microarray technique.In this study, a total of 365 allelic polymorphisms of HLA-DRB genes were searched. A total of 20 sets of primer pairs were used for specific PCR amplification specific for HLA-DRB1, -DRB3, -DRB4 and -DRB5 genes, then hybridized with 124 oligonucleotide probes which were designed to be complimentary to the target olymorphic region of each primer pair. Each 5' primer employed in this study were labeled with fluorescence dye, which can be detected by the scanner of microarray system in the case of successful hybridization with the probe spotted on a glass tip.As a result, 14 sets of primer pairs and 103 of probes adopted to these focus on a Japanese population, were successfully applied to genotyping of 70 genomic DNAs extracted from 20 Japanese homozygous cells and 50 individual. Identity between genotypes of 20 homozygous DNAs typed by this method and those obtained by PCR-RFLP (restriction fragment length polymorphism) were 80%, likewise, 69% for 50 individuals. It will be necessary to modify probe design to increase a accuracy, however, it will become suitable for HLA allele typing if low-background can be attained to prevent false signals of heterozygous.