Yang Dongya Y, Eng Barry, Saunders Shelley R
Department of Archaeology, Simon Fraser University, Burnaby, BC V5A 1S6, Canada.
Hum Biol. 2003 Jun;75(3):355-64. doi: 10.1353/hub.2003.0050.
When highly efficient polymerase was used with high cycle numbers (50-60), strong amplifications were observed, but negative controls were also unexpectedly amplified in a study of ancient human mtDNA from 2000-year-old skeletons. The results of a series of tests revealed that the hypersensitive polymerase chain reaction (PCR) generated by higher cycles and the presence of contaminant DNA (though at extremely low levels) should be responsible for the amplification of negative controls. We suggest that PCR sensitivity be optimized to take advantage of highly efficient polymerase and at the same time prevent "background DNA" from becoming "contaminant DNA" and obscuring the analysis of authentic ancient DNA. We propose the use of multiple positive controls when amplifying ancient human mtDNA samples to indicate the sensitivity of individual PCR amplifications and to monitor the contamination levels of modern human DNA. This study provides some suggestions as to how to amplify and analyze ancient human mtDNA when unavoidable and extremely tiny amounts of modern human DNA exist.
当使用高效聚合酶并进行高循环数（50 - 60）扩增时，观察到了强烈的扩增信号，但在一项对来自2000年前骨骼的古代人类线粒体DNA（mtDNA）研究中，阴性对照也意外出现了扩增。一系列测试结果表明，较高循环数产生的超敏聚合酶链反应（PCR）以及污染DNA的存在（尽管含量极低）是导致阴性对照扩增的原因。我们建议优化PCR灵敏度，以便在利用高效聚合酶的同时，防止“背景DNA”变成“污染DNA”并干扰对真实古代DNA的分析。我们提议在扩增古代人类mtDNA样本时使用多个阳性对照，以表明单个PCR扩增的灵敏度，并监测现代人类DNA的污染水平。本研究就存在不可避免且极其微量的现代人类DNA时如何扩增和分析古代人类mtDNA提供了一些建议。
