'Mitominis': multiplex PCR analysis of reduced size amplicons for compound sequence analysis of the entire mtDNA control region in highly degraded samples
'Mitominis': multiplex PCR analysis of reduced size amplicons for compound sequence analysis of the entire mtDNA control region in highly degraded samples
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DOI：
10.1007/s00414-008-0227-5
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发表时间：
2008-09-01
影响因子：
2.1
通讯作者：
Parson, Walther
中科院分区：
文献类型：
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作者：
Eichmann, Cordula;Parson, Walther
传统的法医线粒体DNA(MtDNA)分析方法包括扩增和测序线粒体DNA控制区的两个高变区。这些引物通常跨越每个区域300-400bp的片段大小，这可能导致在高度降解的样品中扩增较弱或失败。在这里，我们介绍了一种改进的更稳定的方法，使用在144到237bp之间的片段范围内的缩短的扩增片段。需要10个这样的扩增片段才能产生覆盖整个人类mtDNA控制区的重叠片段。在两个多重聚合酶链式反应中对这些基因进行共扩增，并用各自的扩增引物进行测序。我们精心选择了引物，以最大限度地减少与同型和单倍群特异位点的结合，否则会因为错误的引发而导致扩增损失。这种复合体已经成功地应用于古代和法医样本，如显示出高度退化的骨骼和牙齿。
The traditional protocol for forensic mitochondrial DNA (mtDNA) analyses involves the amplification and sequencing of the two hypervariable segments HVS-I and HVS-II of the mtDNA control region. The primers usually span fragment sizes of 300-400 bp each region, which may result in weak or failed amplification in highly degraded samples. Here we introduce an improved and more stable approach using shortened amplicons in the fragment range between 144 and 237 bp. Ten such amplicons were required to produce overlapping fragments that cover the entire human mtDNA control region. These were co-amplified in two multiplex polymerase chain reactions and sequenced with the individual amplification primers. The primers were carefully selected to minimize binding on homoplasic and haplogroup-specific sites that would otherwise result in loss of amplification due to mis-priming. The multiplexes have successfully been applied to ancient and forensic samples such as bones and teeth that showed a high degree of degradation.