应用寡核苷酸芯片进行HLA-DQA1位点基因分型_医学论文.docVIP

应用寡核苷酸芯片进行HLA-DQA1位点基因分型_医学论文 应用寡核苷酸芯片进行HLA-DQA1位点基因分型_医学论文 作者：王彤，王天骄，何群，张玉魁，马佳明，侯伟建， 王绍成，潘忠诚，赵雨杰 【摘要】 为了构建HLA-DQA1位点寡核苷酸分型芯片并籍此建立一种应用于HLA系统基因分型的集成化技术平台，在多态性集中的HLA-DQA1第二外显子区域，自行设计一套寡核苷酸分型探针；采用本实验室自建的方法，制成寡核苷酸芯片。提取的基因组DNA以组间特异性引物，单侧引物荧光标记，行不对称扩增。杂交后扫描检测分析杂交信号，确定HLA等位基因型；分型结果以标准DNA和PCR产物测序检测。结果表明： 100例样本芯片分型全部成功，其中50例标准DNA与其模板相符，另50例临床样本中随机选取的10例与其测序结果相符，其中2例分型结果不完全；重复杂交实验中，位点重现率95%。结论： 研制的HLA-DQA1等位基因分型芯片，其准确性和重现性理想，且操作简便、快捷，有广泛的应用前景。 【关键词】 基因分型　　HLA-DQA1 Genotyping by Using Oligonucleotide Microarrays 　　Abstract In order to fabricate the HLA-DQA1 genotyping chip and develop an integrated，parallel technical platform to type HLA system，a pair of primers and a set of probes were designed according to the sequences of HLA-DQA1 exon 2，where the polymorphism is concentrated. The oligonucleotide chip was made with the methods developed in our laboratory. The target DNA was asymmetrically amplified with the labeled sense primer. The signals were scanned and analyzed after the hybridization between microarray and PCR product. The allele types of the samples were idenlified. The result was verified by the standard DNA and DNA sequencing. The results showed that the genotyping was successfully carried out in 50 standard DNA samples and 50 clinical samples. Among them，results of the 50 standard DNA samples matched their templates. In the other 50 samples，results of the randomly selected 10 matched their sequencing results except that two of them got the incompletely result. In reproducible tests，the signal reappear rate was 95%. It is concluded that HLA-DQA1 genotyping by using our array system is simple and convenient with satisfied accuracy and reproducibility. 　　Key words HLA-DQA1；genotype；oligonucleotide chip 组织配型的情况是影响器官移植成活率的关键因素，同时，特定的HLA基因座位还和某些疾病相关联，因此HLA的分型工作亦受到广泛重视。到目前为止，国内外的HLA的分型研究仍多集中于A、B、DR位点。近年来，组成较为复杂的Ⅱ型位点因与疾病有较多关联而多有报道。其中DQ位点与扩张性心肌症［1］，病毒性乙型肝炎［2］，糖尿病［3，4］，银屑病［5］等多种疾病的罹患率及预后相关。1996年，HLA-DQ也被定义为移植抗原［6］。 目前，HLA分型有血清学分型和基因分型，后者更具优势和发展前途。对基因分型国际上推荐使用PCR-SSP和PC