Identification of DNA of human origin based on amplification of human-specific mitochondrial cytochrome b region
Identification of DNA of human origin based on amplification of human-specific mitochondrial cytochrome b region
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DOI：
10.1016/j.forsciint.2004.07.019
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发表时间：
2005-09-10
影响因子：
2.2
通讯作者：
Yukawa, N
中科院分区：
文献类型：
--
作者：
Matsuda, H;Seo, Y;Yukawa, N
线粒体细胞色素 b 基因非多态性区域中的物种特异性差异似乎足够大，足以允许对法医 DNA 样本进行人类特异性扩增。因此，我们开发了一种基于 PCR 的方法，使用新设计的引物来扩增人类线粒体细胞色素 b 基因的 157 bp 部分。正向和反向引物设计用于与人类线粒体细胞色素 b 基因的区域杂交，其序列与黑猩猩的序列分别相差 26% (7 bp/27 bp) 和 26% (6 bp/23 bp)。使用该引物对，我们成功扩增了从 48 名健康成年人的血液样本中提取的 DNA。所有这些人类样本在琼脂糖凝胶电泳上均产生了预期大小的单条带，并且序列分析表明该单条带的序列与目标区域（157 bp）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、日本猴、食蟹猴）和其他物种（牛、猪、狗、山羊、大鼠、鸡和金枪鱼）的血液样本中提取的 DNA 中没有扩增出可见条带。因此，使用该引物对进行 PCR 扩增后产生单条带的 DNA 可以合理地解释为人类来源。此外，包括血迹、毛干和骨头的老化生物样本被成功鉴定为人类来源，说明了本方法对法医样本的适用性。 (c) 2004 Elsevier Ireland Ltd. 保留所有权利。
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. (c) 2004 Elsevier Ireland Ltd. All rights reserved.