寡核苷酸DNA微阵列用于人类白细胞抗原-DR53组基因分型的研究

Development of oligoneucleotide arrays for HLA-DR53-associated group genotyping

目的 建立用于人类白细胞抗原 (HLA) DR5 3组基因分型的寡核苷酸DNA微阵列。方法 根据中国汉族人群HLA DRB等位基因的频率设计特异性的分型探针 ,制作寡核苷酸DNA微阵列。通过组间特异引物扩增基因组DR5 3相应区段的DNA ,扩增中用Cy5 dCTP荧光标记 ,扩增标记后的产物与芯片的探针杂交 ,通过杂交产生的荧光信号及分布格局确定样品的基因亚型。分型结果用标准DNA和序列特异寡核苷酸探针杂交验证。 结果 经寡核苷酸DNA微阵列分型 ,111份样本中共检出DR5 3组位点 72个 ,其中DR934个 ,DR42 5个 ,DR713个 ,无一例假阳性或假阴性 ,重复率和准确率均达到 10 0 %。检测总耗时约 5h。 结论 寡核苷酸DNA微阵列用于HLA DR5 3组基因分型具有高分辨率、高特异性和简单快速的特点 ,适合于临床应用。

Objective To develop a method performed on an oligoneucleotide array for HLA-DR53 group genotyping. Methods According to the specific allelic frequency and sequence of HLA-DRB loci in Chinese Han population, HLA-DR53 group typing probes which were immobilized on a glass supports were synthesized. A pair of group-special primers labeled by the Cy5-dCTP were designed, and the primers were used in the PCR, thus the PCR products were labeled with Cy5. The labeled PCR products were hybridized with array. The signals were scanned by scanner and analyzed by image software. The typing results were confirmed by standard DNA and PCR-SSO. One hundred and eleven samples were typed by this array. Results There were 72 HLA-DR53 group loci typed by oligoneucleotide array. Among them, 34 loci were DR9, 25 were DR4, and 13 were DR7. No false positive or false negative typing results were observed. The specificity and reproducibility were 100% and the overall time of genotyping was 5 hours.Conclusion The oligoneucleotide array technique is a precise, rapid molecular method for HLA-DR53 genotyping, suited for clinical practice.