Kurihara A, Tawata M, Ikegishi Y, Aida K, Onaya T
Third Department of Internal Medicine, Yamanashi Medical University, Tamaho, Yamanashi-ken, Japan.
Life Sci. 1999;64(14):1223-30. doi: 10.1016/s0024-3205(99)00054-5.
We reported 56 mitochondrial DNA (mtDNA) mutations in Japanese compared with the Cambridge Sequence by polymerase chain reaction-restriction fragment-single strand conformation polymorphism (PCR-RF-SSCP) analysis. Here we report the principle and the detailed procedures of PCR-RF-SSCP analysis. Restriction map of the 15,673 bp PCR product of mtDNA was designed by MacMolly Tetra, and Hha I and Hinc II were selected. Restriction fragment length polymorphism (RFLP) analysis of 15,673 bp PCR products by Hha I/Hinc II revealed one common pattern and 10 polymorphic patterns compared with the prediction based on the Cambridge Sequence. When the samples were electrophoresed by SSCP analysis, DNA fragments of 912 bp, 772 bp, 761 bp, 634 bp, 431 bp, 327 bp, 226 bp and 171 bp showed 6, 4, 5, 4, 5, 7, 2 and 3 polymorphic patterns, respectively. The determinations of nucleotide sequence of these 46 polymorphic patterns revealed 56 mutations compared with the Cambridge Sequence and 7 of these mutations were common in Japanese. Among these, 20 mutations have not been reported before.
我们通过聚合酶链反应-限制性片段-单链构象多态性（PCR-RF-SSCP）分析，报道了与剑桥序列相比，日本人线粒体DNA（mtDNA）的56个突变。在此我们报道PCR-RF-SSCP分析的原理和详细步骤。利用MacMolly Tetra软件设计了15,673 bp的mtDNA PCR产物的限制性图谱，并选择了Hha I和Hinc II。与基于剑桥序列的预测相比，用Hha I/Hinc II对15,673 bp的PCR产物进行限制性片段长度多态性（RFLP）分析，发现一种常见模式和10种多态性模式。当通过SSCP分析对样品进行电泳时，912 bp、772 bp、761 bp、634 bp、431 bp、327 bp、226 bp和171 bp的DNA片段分别显示出6种、4种、5种、4种、5种、7种、2种和3种多态性模式。对这46种多态性模式的核苷酸序列测定显示，与剑桥序列相比有56个突变，其中7个突变在日本人中很常见。其中，20个突变以前未曾报道过。
