HLA-Cw基因测序分型的研究

Study on the genotyping of HLA-Cw locus by sequence based typing method

目的建立HLA-Cw位点测序分型技术,以用于无关供/受者HLA-Cw基因配型等基础应用研究。方法对HLA-Cw位点的第2和第3外显子,设计、合成一对PCR引物和4条测序引物,探索最佳的PCR反应体系和扩增条件,采用PCR引物扩增HLA-Cw基因的第2、第3外显子和第2内含子,扩增产物经纯化后作为测序模板,进行4个测序反应,产物经酒精/EDTA/NaAc法纯化,用ABIPrismTM3100测序仪电泳检测,相关的分析软件分析HLA基因型。检测分型结果的可靠性采用基因型已知的U-CLA国际HLA室间质控的DNA样本进行验证。结果检测6例UCLA国际HLA室间质控的DNA样本,HLA-Cw等位基因型与UCLA公布的结果完全一致。结论本文的HLA-Cw基因测序分型方法准确、可靠,具有广泛的应用前景。

Objective To establish the high-resolution genotyping assay at HLA-Cw locus by sequence based typing （SBT） method, and use the established method in further application of non-related dornor/recipient pair matching. Methods One PCR primer pair and four sequencing primers were designed and synthesized. The concentration of each PCR component and the PCR cycling parameter were adjusted to the optimum so as to amplify a fragment spanning exons 2 and 3 and the interval intron 2 of HLA-Cw gene. The purified PCR product was then utilized as the DNA template in the sequencing reaction, four direct sequencing reaction of PCR product covering exons 2 and 3 in both directions were performed using the ABI Prism BigDye terminator cycle sequencing ready reaction kit, sequencing reaction products purified by EtOH/EDTA/NaAc method were sequenced by ABI Prism 3100 DNA Sequencer. A total of 6 HLA DNA exchange samples distributed by UCLA immunogenetics center were typed and confumed by our SBT method. Results Six international DNA exchange samples genotyped at HLA-Cw locus by above method were completely in accordance with the results provided by UCLA. Conclusion The results indicate that this SBT technique can provide accurate and reliable results at HLA-Cw locus, it shows a broad prospect for its further use in HLA-Cw genotyping.