[Establishment of a large-scale bi-directional sequencing and genotyping platform for MICA gene exons 2 to 4].

Objective To establish a stable and large-scale bi-directional sequencing platform for genotyping MICA gene exons 2 to 4,and to analyze single nucleotide polymorphisms(SNP) of the region.Methods Primers for particular alleles of MICA gene exons 2 to 5 were designed.Optimal conditions for PCR amplification and sequencing reaction were explored.A commercialized one-way sequencing kit for MICA allele was used as a parallel control.Four samples carrying a MICA * 010 allele were subjected to cloning and haplotype sequencing.Results Results of MICA allele typing of 100 samples for a parallel control group were confirmed by the establish method.Twenty-two SNP in MICA gene exons 2 to 4 were detected in Chinese population.Two novel allelic sequences were accepted by GenBank and IMGT/HLA database and officially named as MICA * 065 and MICA * 066 by the WHO Nomenclature Committee.A novel SNP in MICA gene intron 3 was discovered,with allelic sequence submitted to GenBank and IMGT/HLA database.Conclusion The bi-directional sequencing genotyping platform may be applied for largescale study of MICA allelic polymorphisms,tissue typing,organ transplantation and disease research. Key words: Human major histocompatibility complex class Ⅰ chain-related gene A;  Bi-directional sequencing;  Polymorphism;  Genotyping