线粒体多态性检测寡核苷酸芯片的构建

目的构建用于线粒体DNA（mtDNA）D-环控制区（D—LOOP区）多态性多个位点集成检测的寡核苷酸芯片。方法根据GenBank中人mtDNA D—LOOP区序列设计2组引物普通引物和N，N＇-对羧苄基吲哚三菁（Cy5）标记引物]和55种寡核苷酸探针。将探针固定于醛基修饰载玻片表面，制备mtDNA多态性检测芯片。提取40例健康人外周血标本mtDNA，应用Cy5标记引物不对称PCR扩增mtDNA D—LOOP区片段。取未变性和变性后扩增产物分别与芯片进行杂交，观察二者杂交信号强度差异，并将芯片检测结果与DNA测序结果进行比较。观察等倍稀释的不对称PCR扩增产物的杂交信号强度，检测芯片的灵敏度；将DNA测序结果与探针进行BLAST2比对，找出与PCR扩增产物完全匹配和仅1个碱基不匹配的探针，分析二者在芯片上相应位点杂交信号强度的差异，观察芯片的特异性；以放置6个月后的芯片检测外周血mtDNA，观察杂交信号强度的变化。结果不对称PCR扩增获得的mtDNA D—LOOP区片段（466bp）与预期表达片段大小一致。不对称PCR扩增产物直接杂交和变性后杂交的信号强度无明显差异，40例健康人外周血标本mtDNA的芯片杂交检测结果均与DNA测序结果完全吻合。灵敏度检测结果显示，杂交信号强度随扩增产物稀释程度的增加而减弱，芯片检测靶分子的灵敏度为0．1ng；与DNA测序结果完全匹配的探针174和仅相差1个碱基“C”的探针174c的杂交信号强度具有明显差异；而保存6个月后的芯片杂交信号强度基本保持不变。结论构建的寡核苷酸芯片可满足对mtDNA D—LOOP区多态性进行多个位点快速通量检测的需要，具有较高的灵敏度和特异性，适用于法医鉴定及线粒体疾病的检测。

Development of a DNA microarray for detection of mtDNA D-LOOP polymorphism

Objective To develop a high-throughput microarray to identify multiple mtDNA single nucleotide polymorphisms (SNP) sites in D-LOOP region. Methods PCR primers and 55 kinds of oligo-nucleotide probes were designed according to human mtDNA D-LOOP district sequence which was provided by the National Center for Biotechnology Information (NCBI) in the USA. The primers were divided into two groups: one group was labeled with Cy5 while the other was unlabeled. The oligo-nucleotide probes were covalently immobilized on aldehyde modified glass slides to make mtDNA polymorphism microarray. mtDNA was extracted from 40 healthy people’s peripheral blood samples, the mtDNA D-LOOP fragments amplified by asymmetric PCR were add to the microarray after or before denaturalization for hybridization, to observe the difference in the hybridization signal intensity between two groups, and the results of microarray test were compared with those of DNA sequence. The hybridization signal intensity of fold diluted asymmetric PCR amplification products was observed for determining the sensitivity of microarray. The probes completely matched and only one base mismatched with the PCR amplification products were identified after they were compared with the results of DNA sequence by BLAST2, and the difference of the hybridization signal intensity between the two probes was analyzed to observe the specificity of microarray. After storage for 6 months, the microarray was used to detect the mtDNA extracted from peripheral blood, to observe the hybridization signal intensity for detecting the stability of microarray. Results The size of the amplified mtDNA D-LOOP fragment (466 bp) was same to that of the expected DNA fragment. There was no significant difference in the hybridization signal intensity of asymmetric PCR amplification products between direct hybridization and hybridization after degeneration, the results of mtDNA polymorphism detected by microarray from 40 healthy people’s peripheral blood samples were consistent with DNA sequence. The sensitivity experiment showed that the hybridization signal intensity was decreased with diluted fold increasing, and the sensitivity limit of microarray was 0.1 ng. There was significant difference in the hybridization signal intensity between the probe 174 completely matched and the probe 174c only one base “C” mismatched with the PCR amplification products compared with the results of DNA sequence. The hybridization signal intensity was almost constant after storage for 6 months. Conclusions The DNA microarray could meet the need of the quick through-out multiplex detection of the point mutations in mtDNA D-LOOP polymorphism. It has high sensitivity and specificity, and can be used for forensic medicine identification and diagnosis of mtDNA mutation related disorders.