HLAⅠ类抗原的基因芯片分型技术研究

DNA typing of HLA-A,B antigens by DNA chip technique

目的 采用DNA芯片技术进行汉族人群HLAⅠ类抗原A、B位点的DNA分型研究 ,并实现将A、B位点的DNA分型集中于一张芯片上 ,同时完成。方法 根据HLAⅠ类抗原A、B位点不同基因亚型的独特序列设计探针 ,制成分型芯片 ;待检测样品经PCR反应标记上荧光之后 ,与探针在芯片上进行杂交 ,通过对杂交产生的荧光信号值进行分析 ,确定样品的HLA A、B基因亚型。将这一方法应用于 2 2 0份样本的HLAⅠ类DNA分型并将部分样品进行基因测序。结果 所有样本的HLAⅠ类基因芯片分型均获得成功 ,无假阳性和假阴性出现 ,80份样本的重复率为 10 0 % ,总耗时 2 .5h。分型结果经双盲验证完全符合。可准确分辨出A位点等位基因 2 0个 ,B位点等位基因 4 1个 ,实际检出汉族人群A抗原特异性 13个、B抗原特异性 32个。结论 基因芯片用于HLAⅠ类A、B分型技术可行 ,其分辨率高、特异性强、重复性好、操作简便快速、结果直观 ,可以在一张芯片上同时检测HLA A、B位点 ,并实现一张芯片多人份 ,适合于临床应用。

Objective To establish a DNA typing method for HLA-A, B antigens by DNA chip technique and simultaneous typing HLA-A, B alleles in one chip. Methods According to the sequence of HLA-A and HLA-B alleles, chip was made, then labeled PCR products hybridized with them, the signals were scanned by scanner 4?000 and analyzed by Imagene software. We have genotyped 220 samples and sequenced some of them. Results HLA-A, B alleles were successfully typed in 220 clinical samples and 62 standard DNAs by DNA chip technique. No false positive or false negative typing results were recorded. Reproducibility was 100% in 80 samples. The overall time of DNA typing was 2.5 hours. Twenty alleles of HLA-A and forty-one HLA-B alleles were accurately differentiated. Thirteen HLA-A antigen specificity and thirty-two HLA-B antigen specificity in Chinese were typed. Conclusion DNA typing for HLA-A, B alleles by DNA chip has been proved to be a technique of high-resolution, high-specificity and reproducibility, and it is more intuitional and more suitable for clinical application than serological technique.