非放射性反相杂交法在HLA－DQA1位点基因分型中的应用

Application of Non-radioactive Reverse Dot Blot Hybridization to HLA-DQA1 Genotyping

将遴选的经适当接尾的１２个ＨＬＡ－ＤＱＡ１序列特异性寡核苷酸固定在一张滤膜上，用生物素标记的ＤＱＡ１特异性扩增产物与滤膜上的序列特异性寡核苷酸在四甲基氯化铵杂交体系中杂交，然后经洗膜封膜，杂交信号用非放射性的碱性磷酸酶显色法检测，根据杂交斑点的显示结果分析标本的基因型。采用这种方法初步确定了ＨＬＡ－ＤＱＡ１位点８种单倍型等位基因：ＤＱＡ１０１０１、０１０２、０１０３、０２０１、０３０１１、０４０１、０５０１和０６０１．非放射性反相杂交法可对各种来源的杂合性标本进行ＨＬＡ－Ⅱ类基因快速分型，并适合在临床器官移植的组织分型配型、疾病易感性研究和法医鉴定等领城中应用。

Twelve sequence-specific oligonucleotide of HLA-DQAl were dotted on a nylon filter.Biotinated PCR products were hybridized with 12 SSO. Hybridization signals was detected by color development of alkaline phosphotase catalysing BCIP/NBT substrate reaction. HLA-DQA1 genotyping of clinical samples were made by this method,and primarily determined 8 genotypes,i. e. DQA10101, 0102, 0103, 0201, 03011, 0401, 0501 and 0601. It provided a practical method for rapid genotyping of various heterozygous samples, tissue matching of clinical transplantation,study of diseases sensrtivity and forensic medicine identification.