Reliable nuclear and mitochondrial DNA quantification for low copy number and degraded forensic samples
Reliable nuclear and mitochondrial DNA quantification for low copy number and degraded forensic samples
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DOI：
10.1016/j.fsigss.2011.09.014
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发表时间：
2011-12-01
期刊：
影响因子：
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通讯作者：
Pestano, J.
中科院分区：
文献类型：
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作者：
Fregel, R.;Almeida, M.;Pestano, J.
DNA定量是低拷贝数法医分析和古DNA研究的先决条件。此外，如果核定量集中在淀粉原基因位点，它也允许性别决定。这些技术的一些问题是，由于人类种内变异影响引物和/或探针的退火，在淀粉原基因位点和线粒体DNA （mtDNA）定量偏差的等位基因脱落现象。本文提出的方法结合了两种多重TaqMan (R)实时PCR (qPCR)，用于降解或有限样品的核和mtDNA定量。核DNA检测是基于X和Y染色体特异性片段在淀粉原基因位点的独立扩增和内部PCR控制（IPC）来识别抑制问题。片段长度较短（71 bp）有利于对严重降解的DNA进行定量分析，而在LCN分析中，使用两组不同的引物进行X和Y染色体扩增是为了减少等位基因缺失。MtDNA的定量是基于位于MtDNA 16S区的三个PCR片段的扩增。其中两种是用人类特异性保守引物和探针扩增的，这使得该技术在世界范围内的应用成为可能。此外，它们的长度差异（分别为167和314 bp）提供了DNA降解水平的信息。为了识别非人类DNA，还设计了一个种间mtDNA片段（187bp）。2011爱思唯尔爱尔兰有限公司版权所有。
DNA quantification is a prerequisite for both low copy number (LCN) forensic analysis and ancient DNA (aDNA) studies. Moreover, if nuclear quantification is focused on the amelogenin locus, it also allows sex determination. Some of the problems of these techniques are allelic drop-out phenomenon in amelogenin locus and mitochondrial DNA (mtDNA) quantification biases, due to human intraspecific variation affecting the annealing of primers and/or probes.The method presented here combines two multiplex TaqMan (R) real-time PCR (qPCR) for nuclear and mtDNA quantification in degraded or limited samples. Nuclear DNA detection is based on the independent amplification of X and Y chromosome specific fragments in the amelogenin locus and an internal PCR control (IPC) to recognize inhibition problems. The small length of the fragments (71 bp) favors the quantification of severely degraded DNA, whereas the use of two distinct primer sets for X and Y chromosome amplification is directed to reduce allelic drop-out in LCN analysis. MtDNA quantification is based on the amplification of three PCR fragments located in the mtDNA 16S region. Two of them are amplified with human specific conservative primers and probes, which allows a world-wide application of this technique. Moreover, their length difference (167 and 314 bp respectively), provides information about the DNA degradation level. In order to also recognize non-human DNA an interspecific mtDNA fragment (187 bp) was also designed. (C) 2011 Elsevier Ireland Ltd. All rights reserved.