Grzybowski Tomasz, Malyarchuk Boris A, Czarny Jakub, Miścicka-Sliwka Danuta, Kotzbach Roman
The Ludwik Rydygier Medical University, Forensic Medicine Institute, Bydgoszcz, Poland.
Electrophoresis. 2003 Apr;24(7-8):1159-65. doi: 10.1002/elps.200390149.
The present study demonstrates a reinvestigation of the mitochondrial DNA sequence heteroplasmy, which was previously found by the use of nested polymerase chain reaction (PCR) technique in single hairs of 13 individuals. The direct PCR approach was used for the amplification of mitochondrial DNA and a phylogenetic analysis was applied to both data sets for the verification of the authenticity of sequences. The comparative analysis of the sequencing results obtained from the same hair DNA extracts - but using two different techniques - shows that direct mitochondrial DNA amplification results in a considerably lower number of mixed positions. The majority of the confirmed heteroplasmic variants preferentially occurs in mitochondrial DNA hypervariable sites (mutational hotspots). However, the pattern of heteroplasmic mutations observed in four extracts after nested PCR significantly differs from the pattern of natural mutations. Some of these rare polymorphisms should be revised as inconsistent with phylogenetic expectations. The results of the present study contribute to the earlier reports by indicating that phylogenetic analysis is an effective tool in a posteriori quality check of mitochondrial DNA data.
本研究展示了对线粒体DNA序列异质性的重新调查，该异质性先前是通过巢式聚合酶链反应（PCR）技术在13个人的单根毛发中发现的。直接PCR方法用于扩增线粒体DNA，并对两个数据集进行系统发育分析以验证序列的真实性。对从同一毛发DNA提取物中获得的测序结果进行比较分析——但使用两种不同技术——表明直接线粒体DNA扩增导致的混合位置数量要低得多。大多数已确认的异质变体优先出现在线粒体DNA高变区（突变热点）。然而，巢式PCR后在四种提取物中观察到的异质突变模式与自然突变模式显著不同。其中一些罕见的多态性应根据系统发育预期进行修正。本研究结果通过表明系统发育分析是线粒体DNA数据事后质量检查的有效工具，为早期报告做出了贡献。
