特异性寡核苷酸探针反向杂交在mtDNA点突变检测中的应用研究

目的：建立一种快捷、有效的mtDNA点突变检测体系。方法：线粒体病患者15例，根据临床表现分为3组，线粒体脑肌病伴高乳酸血症、卒中样发作（MELAS）组7例，单纯型线粒体肌病组4例，慢性进行性眼外肌麻痹（CPEO）组4例。用苯酚／氯仿法提取外周全血DNA，采用PCR／ASO反向杂交技术。用PCR扩增mtDNA的突变“热区”——tRNA^Leu（UUR）基因，在下游引物的5’端标记上地高辛，与点在尼龙膜上的5对寡核苷酸（AS0）探针：A3243／A3243G（野生型／突变型）、T3271／T3271C、G3460／G3460A、T3291／T3291C、C3256／C3256T进行反向杂交，根据杂交显色信号，确定有无突变。同时利用PCR／RFLP方法加以验证。结果：MELAS组中有A3243G点突变3例，但没有其他4个位点的突变。单纯型线粒体肌病组和CPEO组均未发现以上5个位点的突变。PCR/ASO反向杂交法与PCR／RFLP法结果一致。结论：PCR/ASO反向杂交技术，适用于已知mtDNA点突变的检测，具有敏感、节时、节省费用的特点。尤其适用于大批量标本的筛选。

Reverse dot-blot hybridization with allele-specific oligonucleotide probes in detection of mtDNA point mutations

Objective: To establish a speedy, efficient detective method and a system of mtDNA mutation. Methods: Fifteen cases were divided according to their clinical characteristics: 7 cases of MELAS, 4 cases of pure mitochondrial myopathy, and 4 cases of CPEO. Genomic DNA was extracted from peripheral blood with phenol/chloroform procedure. PCR/ASO reverse dot-blot hybridization was used to perform PCR-amplification at the mutation hot spot-tRNALeu(UUR) region, and reverse primer was marked by Digitalin at 5' end. Then the products of PCR were hybridized with 5 pairs of ASO probe A3243/A3243G(wild type/mutation type), T3271/T3271C, G3460/G3460A, T3291/T3291C, C3256/C3256T which were spotted on the nylon membrane. Being recognized the hybridization signal, the results were determined whether the PCR products had mutations or not, and then PCR products were tested and verified with PCR/RFLP. Result: In MELAS group, 3 cases had A3243G point mutation, but not another 4 point mutations. In pure mitochondrial myopathy and CPEO group, none had the 5 point mutations. The results were consistent between PCR/ASO and PCR/RFLP. Conclusion: PCR/ASO reverse dot-blot hybridization can be widely applied to detect the known mtDNA point mutations with the advantages of sensitivity, effect and economy. The procedure is applicable for mass samples detection especially.