Matsuda Hirokazu, Seo Yasuhisa, Kakizaki Eiji, Kozawa Shuji, Muraoka Eri, Yukawa Nobuhiro
Department of Legal Medicine, Miyazaki Medical College, 5200 Kihara, Kiyotake-cho, Miyazaki 889-1692, Japan.
Forensic Sci Int. 2005 Sep 10;152(2-3):109-14. doi: 10.1016/j.forsciint.2004.07.019.
Species-specific differences in a non-polymorphic region of the mitochondrial cytochrome b gene appear to be large enough to allow human-specific amplification of forensic DNA samples. We therefore developed a PCR-based method using newly designed primers to amplify a 157-bp portion of the human mitochondrial cytochrome b gene. The forward and reverse primers were designed to hybridize to regions of the human mitochondrial cytochrome b gene with sequences differing from those of chimpanzee by 26% (7 bp/27 bp) and 26% (6 bp/23 bp), respectively. Using this primer pair, we successfully amplified DNA extracted from blood samples of 48 healthy adults. All these human samples produced a single band of the expected size on agarose gel electrophoresis, and the sequence of the single band was shown to be identical to that of the target region (157 bp) by sequence analysis. On the other hand, no visible bands were amplified from DNA extracted from blood samples of animals including non-human primates (chimpanzee, gorilla, Japanese monkey, crab-eating monkey) and other species (cow, pig, dog, goat, rat, chicken and tuna). Thus, DNA producing a single band following PCR amplification using this primer pair can be reasonably interpreted as being of human origin. In addition, aged biological specimens comprising bloodstains, hair shafts and bones were successfully identified as being of human origin, illustrating the applicability of the present method to forensic specimens.
线粒体细胞色素b基因非多态性区域的物种特异性差异似乎足够大，足以实现法医DNA样本的人类特异性扩增。因此，我们开发了一种基于PCR的方法，使用新设计的引物扩增人线粒体细胞色素b基因的157bp片段。正向和反向引物设计为分别与人类线粒体细胞色素b基因中与黑猩猩序列差异为26%（7bp/27bp）和26%（6bp/23bp）的区域杂交。使用这对引物，我们成功扩增了从48名健康成年人血液样本中提取的DNA。所有这些人类样本在琼脂糖凝胶电泳上均产生了一条预期大小的条带，通过序列分析表明该单一条带的序列与目标区域（157bp）的序列相同。另一方面，从包括非人类灵长类动物（黑猩猩、大猩猩、日本猕猴、食蟹猴）和其他物种（牛、猪、狗、山羊、大鼠、鸡和金枪鱼）的动物血液样本中提取的DNA未扩增出可见条带。因此，使用这对引物进行PCR扩增后产生单一条带的DNA可以合理地解释为人类来源。此外，包括血迹、毛发和骨骼在内的陈旧生物标本也成功鉴定为人类来源，这说明了本方法在法医标本中的适用性。
