摘要
本文介绍一种适合于多等位基因定型和结构分析的反相顺序特异性寡核苷酸探针杂交方法。将化学合成的多种探针分别经脱氧核苷末端转移酶(TdT)催化,于3′端加上100个以上碱基的Poly(dT)尾,然后将各探针分别以斑点固定于同一张尼龙膜上。用PCR法对该基因位点进行扩增,扩增的同时加入α-^(32)P-dCTP以直接参入放射标记,然后将PCR产物与膜固定探针杂交,以四甲基氯化铵根据探针长度统一洗涤温度。该方法克服了以往对同一份样品进行多个等位基因检测时需要进行多次探针标记和杂交的缺点。我们用该方法对中国人群MHC-Ⅱ类DR4多等位基因进行了研究。
A reverse SSO (sequence specific oligonucleotide) probe hybridization method for multiallele typing and structure analysis has been established. The oligonucleotide probes are tailed with poly(dT) by terminal deoxynucleotidyl transferase and then immobilized on a nylon membrane, α-^(32)P-dCTP was added during in vitro amplification of the HLA-DR4 gene so the PCR products were ^(32)P labelled. Aftor hybridization, the membrane was washed with tetramethyl ammonium chloride solution, the temperature was controled according to the length of the probes. The method obviated the repeated probe labelling and hybridization for multiallele testing of one sample. We have used this method to analyze MHC-class Ⅱ DR4 gene subtypes in Chinese population.
基金
国家自然科学基金
关键词
反相杂交法
多等位基因
检测
Reverse hybridization
Multiallele analysis
HLA DR4
