Tobe Shanan S, Linacre Adrian M T
Centre for Forensic Science, WestCHEM, Department of Pure and Applied Chemistry, University of Strathclyde, Glasgow, UK.
Forensic Sci Int Genet. 2008 Sep;2(4):249-56. doi: 10.1016/j.fsigen.2008.03.002. Epub 2008 May 2.
The number of mitochondria per cell varies by cell type and the number of mitochondrial DNA (mtDNA) genomes varies per mitochondrion. Biological samples from unknown species are encountered frequently in forensic science investigations and are often contaminated with human mtDNA making analysis difficult. Currently, no techniques to quantify non-human mtDNA are available. We report on a method to accurately quantify, sensitive to 100 copies (1.7fg), mtDNA from human and non-human sources when present as a mixture. The test developed uses the cytochrome b (cytb) and the ribosomal 12S genes on the mitochondrial genome. Universal and human specific fragments of similar size are amplified and quantified using SYBR Green. We validate the test with 24 human samples and 27 non-human mammalian samples. The human fraction of a sample can then be subtracted from the universal fraction for an accurate estimation of non-human mtDNA copy number.
每个细胞中的线粒体数量因细胞类型而异，每个线粒体中的线粒体DNA（mtDNA）基因组数量也有所不同。在法医学调查中，经常会遇到来自未知物种的生物样本，并且这些样本常常被人类mtDNA污染，这使得分析变得困难。目前，尚无定量非人类mtDNA的技术。我们报告了一种方法，该方法能够准确地定量存在于混合物中的来自人类和非人类来源的mtDNA，灵敏度可达100个拷贝（1.7fg）。所开发的检测方法利用线粒体基因组上的细胞色素b（cytb）和核糖体12S基因。使用SYBR Green对大小相似的通用片段和人类特异性片段进行扩增和定量。我们用24个人类样本和27个非人类哺乳动物样本对该检测方法进行了验证。然后，可以从通用部分中减去样本的人类部分，以准确估计非人类mtDNA的拷贝数。
