基因芯片在中国南方汉族人白细胞抗原-DRB分型中的应用  

Genotyping of HLA-DRB by oligonucleotide arrays in Han populations in southern China

作　　者：肖家全[1] 谭建明[1] 李成涛 康敏华 方燕红 李瑶 沈瑾[1] 丁言德[1] 

机构地区：[1]上海市第一人民医院,上海市器官移植研究中心,200080 [2]上海联合基因科技集团,博星芯片研究所

出　　处：《中华医学杂志》2003年第5期417-420,共4页National Medical Journal of China

基　　金：全军"十五"大课题杰出人才基金资助项目 ( 0 1J0 0 3);上海市科委科技攻关课题资助项目 ( 0 2 49190 0 5 )

摘　　要：目的　建立一种用于HLA -DRB分型的基因芯片 ,并通过与PCR SSP比较 ,评价芯片的性能。方法　根据中国汉族南方人常见的HLA DRB位点基因及其多态性的独特序列 ,在第二外显子区域设计特异性的寡核苷酸分型探针 ,将其点在玻片上 ,制成芯片。基因组DNA通过组间特异引物扩增 ,扩增中同时用荧光素Cy5标记。扩增标记后的产物与结合在芯片上的探针进行杂交 ,通过荧光扫描仪对杂交产生的荧光信号值进行分析 ,确定样品的HLA DRB基因亚型。将这一方法应用于 110份样本的HLA DRB基因分型并与PCR SSP分型的结果进行比较。结果　 110份淋巴细胞样本 ,除1份PCR无产物致芯片不能分型外 ,其余样本芯片分型全部成功。不吻合样本 10份 :其中SSP定型纯合子 6份 ,芯片分型全部为杂合子 ,SSP定型为杂合子 1份 ,芯片结果为纯合。经第三方用PCR SSO验证 ,证实芯片分型正确。另外 3份不吻合的样本经测序 ,证实SSP分型错误 2份 ,芯片分型错误 1份。芯片的重复率为 95 %。结论　基因芯片是一种理想的分型方法 ,具有特异性高、重复性好、操作简便、所需样本量少、结果判读容易、一次可作多份样本的优点。Objective To develop an oligoneucleotide array for HLA-DRB typing and evaluate its function in comparison with that of PCR-SSP typing. Methods According to the specific allele sequences of HLA-DRB loci in Han populations in Southern China, 44 synthesized typing probes were immobilized on a glass supports. A pair of group-specific primers was designed according to the sequence of HLA-DRB exon2, then the primers and Cy5-dCTP were used in PCR, thus the PCR products were labeled with Cy5. The labeled PCR products were hybridized with the probes in the array, and the signals were scanned by scanner and then analyzed by Image software.110 samples of DNA of the lymphocytes from the spleens or peripheral blood of kidney recipients and unrelated donors were typed by this array and the results were compared with those of PCR-SSP typing. Results All the samples except for one without PCR product had been genotyped by HLA array successfully. Ten samples were identified differently by these 2 methods. PCR-SSO verified the correctness of the array in 7 samples among which 6 samples were identified as homozygous by PCR-SSP and heterozygous by array and 1 sample was identified as heterozygous by PCR-SSP and homozygous by the array; and proved that among the remaining 3 samples the results of 2 samples identified by PCR-SSP and 1 sample identified by the array were wrong. Conclusion The HLA-DRB oligoneucleotide array technique is a precise, rapid molecular method for HLA-DRB genotyping. Compared with PCR-SSP method, the genotyping chip is more sensitive and specific and can test several samples at a time.

关 键 词：基因芯片 中国 南方 汉族人 白细胞抗原-DRB分型 应用 基因分型 器官移植 

分 类 号：R346[医药卫生—基础医学]