地高辛配基标记DNA探针检测HLAⅡ类基因技术的建立

Oligo-Typing with Non-Radioactive Assays for HLA Class Ⅱ Allelic Polymorphism

本文报道了采用序列特异性寡核苷酸探针杂交(SSOPH)检测HLAⅡ类基因(DRB、DQB)多态性的方法。实验包括少量全血提取模板DNA、PCR基因扩增基因多态性区域片断、尼龙膜点样、特异性寡核苷酸探针合成及地高辛配基(DIG-11-ddUTP)标记、探针杂交;条件洗涤、化学发光显影等一系列步骤。该实验方法具有用血量少、结果精确稳定、操作简便安全、不污染环境等优点。作者对模板DNA的提取、杂交缓冲液制备、标记探针的使用剂量以及条件洗涤等多个环节作了改良,使实验方法和结果进一步优化。

In this article, we described a method using sequence - specific oligonucleotide probe hyhridization (SSOPH) to detect the polymorphism of HLA class Ⅱ alleles. The experiment involves a series of procedures;such as DNA extraction,PCR amplification,dot blotting,synthesis of specific oligonucleotide probes, nonradioactive labeling (digoxygenin - Ⅱ -ddUTP), probe hybridization, stringent washing and chemi - Luminescent detection. This method needs only a small amount of peripheral blood. It is sensitive,safe,and easy to handle. The results are quite accurate and stable. A lot of improvment have been done among the whole procedures,so as to optimize the experiment and the results.