A pyrosequencingª²based method for high resolution HLAª²DRB genotyping

    YUAN Jianª²Lin, WU Guoª²Jun, XUE Li, ZHAO Jinª²Rong, BAI Yuª²Jie, YANG Fang2, WANG He, ZHANG Yunª²Tao, YANG Liª²Jun

    (1Department of Urology, Xijing Hospital, 2Institute of Gene Diagnosis, Fourth Military Medical University, Xi¬ðan 710033, China)

    ¡¾Abstract¡¿ AIM: To develop a pyrosequencingª²based typing (PSBT) approach for high resolution identification of HLAª²DRB alleles. METHODS: The DNA fragments from HLAª²DRB exonª²2 were obtained using alleleª²specific or multiplex PCR amplification. The PSBT of purified DNA was performed. RESULTS: The polymorphic residues of HLAª²DRB genes were identified in each pyrosequencing reaction and read length up to 90 nucleotides was obtained. A blood sample containing heterozygous alleles (DRB1ª²0405 and DRB3ª²01011) was analyzed using purified DNA respectively from alleleª²specific or multiplex PCR reaction, the results of which were consistent with those from the traditional method. CONCLUSION: Pyrosequencing used in the analysis of HLAª²DRB alleles has the advantage of high resolution and can be widely employed clinically in donor/recipient selection. ......