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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 establish a gene chip for HLA-DRB typing and evaluate the performance of the chip by comparing with PCR SSP. Methods Based on the unique HLA DRB locus genes and unique sequences of polymorphisms in Chinese Han southerners, a specific oligonucleotide probe was designed in the second exon region, Into the chip. Genomic DNA is amplified by inter-group specific primers that are simultaneously labeled with fluorescein Cy5. After amplification, the labeled product is hybridized with the probe bound to the chip. The fluorescent signal value generated by the hybridization is analyzed by a fluorescence scanner to determine the HLA DRB gene subtype of the sample. This method was applied to HLA DRB genotyping of 110 samples and compared with the results of PCR SSP typing. Results 110 samples of lymphocytes, except for 1 PCR-free product caused by the chip can not be typed, the remaining sample chip all successful. Do not match the sample of 10: SSP Homozygous stereotypes 6, all the chip type heterozygotes, SSP stereotypes as a heterozygote, the chip results for the homozygous. After the third party using PCR SSO verification, confirm the correct chip type. The other 3 samples that did not match were sequenced and confirmed 2 copies of SSP typing errors and 1 chip typing error. The chip’s repetition rate is 95%. Conclusion Gene chip is an ideal typing method. It has the advantages of high specificity, good repeatability, simple operation, small sample size, easy interpretation of results and multiple samples at one time.