MICA基因第2～4外显子大规模双向测序及分子克隆检测平台的建立  

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

作　　者：高素青[1] 邓志辉[1] 徐筠娉[1] 王大明[1] 何柳媚[1] 金士正[1] 

机构地区：[1]广东省深圳市血液中心深圳组织相容性和免疫遗传学重点实验室,518035

出　　处：《中华医学遗传学杂志》2012年第5期542-546,共5页Chinese Journal of Medical Genetics

基　　金：深圳市科技计划项目（200902119）

摘　　要：目的建立稳定的、大规模的MICA基因第2～4外显子双向测序分型检测技术平台，并分析其单核苷酸多态性（single nueleotide polymorphism，SNP）。方法自行设计MICA基因第2～5外显子扩增引物及测序引物，探索PCR扩增及测序的最佳反应条件。用商品化的MICA基因单向测序分型试剂盒作为平行对照，对4个包含MICA＊010等位基因的样本采用自行设计引物扩增，并进行分子克隆和单倍体测序。结果采用自行建立的MICA基因双向测序分型方法验证了100人份平行对照组单向测序分型结果。应用本研究建立的方法获得了中国人群MICA基因第2～4外显子22个SNP位点。两个新等位基因MICA＊065、MICA＊066获得了世界卫生组织的正式命名。首次发现了MICA等位基因第3内含子新的SNP位点，序列已提交IMGT／HLA数据库。结论建立的MICA基因双向测序方法可大规模应用于中国人群的MICA基因多态性、组织器官移植配型和疾病研究。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 large- scale study of MICA allelic polymorphisms, tissue typing, organ transplantation and disease research.

关 键 词：人类主要组织相容性复合体I类相关基因A 双向测序 多态性 基因分型 

分 类 号：R440[医药卫生—诊断学]