人类白细胞Ⅰ、Ⅱ类抗原的中分辨度基因芯片分型技术研究

DNA typing of HLA-ⅠandⅡantigens with medium resolution method by DNA chip technique

目的应用基因芯片技术进行北方汉族人群人类白细胞抗原(HLA)Ⅰ、Ⅱ类中分辨度分型研究。方法根据中国北方汉族人群HLA-Ⅰ、Ⅱ类常见的基因位点以及临床应用中对分型分辨度的实际需要,设计特异性寡核苷酸中分辨度分型探针,制成基因分型芯片。采用带荧光标记的引物,用合适的PCR方法扩增HLA-Ⅰ、Ⅱ类抗原上的多态性区域,产物与芯片上探针杂交。杂交结果经荧光扫描并用特定软件分析判断阳性探针,以此确定样品基因型。共检测30份临床样本。结果所有样本的HLA-Ⅰ、Ⅱ类抗原基因分型均获成功。此中分辨度探针可分辨出598个Ⅰ类抗原等位基因,511个Ⅱ类抗原等位基因,可捡出Ⅰ抗原特异性57个,Ⅱ抗原特异性30个。结论基因芯片用于HLA-Ⅰ、Ⅱ类抗原分型可行。

Objective To establish a DNA typing method for the HLA- Ⅰ and Ⅱ antigens in the Chinese of Han Nationality in Northern China with medium resolution method by DNA chip technique. Methods Corresponding primers for the sense exon fragments of HLA-A, HLA-B, HLA-DR, HLA-DQB, and HLA-DQA were designed. A DNA typing chip was made with specific medium resolution typing probes designed according to the gene frequency of HLA- Ⅰ and Ⅱ alleles among the Chinese of Han nationality in Northern China. Unsymmetrical PCR was used to amplify the HLA-Ⅰ and Ⅱ exon2 and exon3 in 30 samples from the Chinese of Han nationality in Northern China, and then the PCR products were labeled and hybridized with the probes on the chip. The typing of HLA-Ⅰ and Ⅱ was certified by scanning the hybridized products and analyzing them with a set of computer software. Results HLA- Ⅰ and Ⅱ alleles were successfully typed in 30 clinical samples . The h probes with medium resolution were able to discern 42 HLA-Ⅰ and Ⅱ alleles accurately. By using this chip 30 HLA- Ⅰ and Ⅱ alleles were distinguish and 6 new specific alleles were found. Conclusion DNA typing of HLA-Ⅰ and Ⅱ by chip has been proven to be a high-resolution and high-specificity method. It is able to check out the new alleles that can not be distinguished by other methods with the same resolution, and it is more intuitional and more suitable for clinical application.