膜芯片检测糖尿病相关线粒体基因突变位点  

Development of a nylon membrane chip for rapid detecting the diabetes-associated 45 mutant loci in mitochondrial DNA

作　　者：刘松梅[1] 周新[1] 秦汉[2] 刘兵[3] 涂建成[1] 郑芳[1] 李霞[1] 

机构地区：[1]武汉大学中南医院基因诊断中心,430071 [2]黄石理工学院医学院 [3]武汉大学黄石中心医院

出　　处：《中华检验医学杂志》2007年第12期1344-1348,共5页Chinese Journal of Laboratory Medicine

基　　金：国家自然科学基金(30672012);湖北省科技攻关计划资助项目(2006AA301C07)

摘　　要：目的构建一种快速、系统检测与糖尿病相关的45个 mtDNA 位点的膜芯片。方法针对 mtDNA 的45个突变位点,采用 Primer Premier 5.0和 NCBI BLAST 软件设计野生型和突变型探针;将带有8T 长臂的90条探针,通过紫外交联固定于经5×SSC Buffer 处理的尼龙膜;不对称 PCR 方法制备单链靶序列,纯化后经 Biotin 标记,AP-avidin 作用于 NBT/BCIP 显色,目视化观察结果。采用该膜芯片检测24份已知 mtDNA 序列的糖尿病标本,每份标本平行检测3次,评价检测结果。结果 9对引物可在同一条件下特异性扩增45个基因位点的目的片段;野生型和突变型探针的退火温度分别为(59.01±1.42)℃、(59.34±1.29)℃,能够在相同的温度下完成杂交反应;膜芯片样点清晰、规整度好,分布均匀,无漏点和连点,阳性对照和阴性对照结果理想,阴性对照的信号强度与背景持同;除16189位点外,检测结果与 DNA 测序方法有很好的一致性(x^2=113.132,Kappa 值=0.888,P=0.000)。结论成功构建可一次性检测目前国内外报道与糖尿病相关的44个 mtDNA 突变位点的膜芯片,适用于糖尿病患者和高危人群的检测。Objective To develop a nylon membrane chip for rapid and systematic detection of the diabetes-associated 45 mutant loci in mitochondrial DNA （mtDNA）. Methods The mutant- and wild-type probes were designed for detection of 45 mutant loci in mtDNA with Primer Premier 5.0 and NCBI BLAST softwares and the 90 probes with 8 poly T were immobilized on the Hybond N ＋ nylon membranes which were treated with 5 × SSC Buffer by UV-crosslinking; Then asymmetric PCR was employed to obtain the target single strand DNA （ssDNA）. The PCR products were labeled with biotin after purification. NBT/BCIP was used as substrate that yields a very intense purple signal followed by AP-avidin, and the signals were observed in 24 samples with known sequences to evaluate the chips, each sample was repeatedly measured three times. Results The specific target fragments of 45 loci can be amplified under the same condition with nine sets of primers. The annealing temperatures of the wild-type [ （59.01 ± 1.42）℃ ] and mutant-type [ （59. 34 ± 1.29）℃ ] probes are so close （t = 1. 046,P =0. 301 ） that hybridization can be performed at the same temperature. The spots on the membrane chip are distinct, regular and well-distributed. The results of positive- and negative-control are perfect. The signals of negative probes and the background are similar. The results of chip were nearly concordant with that of DNA sequences （X^2 = 113. 132, Kappa value = 0. 888, P = 0. 000） except for T16189C mutant. Conclusions We have successfully developed a nylon membrane chip for rapid and systematic detection of the diabetes-associated 44 mutant loci in mtDNA. It could be used for screening for diabetic patients and high-risk people.

关 键 词：基因芯片 糖尿病 DNA探针 

分 类 号：R587.1[医药卫生—内分泌] R450[医药卫生—内科学]