Development and validation of a D-loop mtDNA SNP assay for the screening of specimens in forensic casework
Development and validation of a D-loop mtDNA SNP assay for the screening of specimens in forensic casework
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DOI：
10.1016/j.fsigen.2013.02.005
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发表时间：
2013-05-01
影响因子：
3.1
通讯作者：
Fagundes, Paulo Roberto
中科院分区：
文献类型：
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作者：
Chemale, Gustavo;Paneto, Greiciane Gaburro;Fagundes, Paulo Roberto
线粒体DNA（mtDNA）分析通常是常规法医DNA案例工作的最后手段。然而，它已成为分析高度降解样品或含有太少或不含核DNA的样品（如旧骨和毛干）的有力工具。金标准方法仍然是对聚合酶链反应（PCR）产物或HVS-1和HVS-2（高变区段）控制区区段克隆的扩增子进行直接测序。使用mtDNA进行鉴定是耗时的，昂贵的，并且可能非常复杂，这取决于被测试材料的数量和性质。这项工作的主要目标是开发一种劳动密集度较低，成本较低的mtDNA分析筛选方法，以帮助排除不匹配的样本，并作为最终确认DNA测序之前的假定测试。基于Salas和Amigo（2010）[1]进行的模拟，我们选择了14种高度区分的单核苷酸多态性（SNP），使用SNaPShot（TM）（Applied Biosystems，Foster City，CA，USA）进行分型。通过对100多个HVS-1/HVS-2测序样品进行分型，验证了该测定法。当比较结果时，除了在少数单倍型中观察到等位基因缺失外，SNP分型和DNA测序之间没有观察到差异。使用代表巴西人群的172个mtDNA序列进行单倍型多样性模拟，当使用14个SNP时获得0.9794的分数，表明Salas和Amigo（2010）[1]提出的选择高度歧视性SNP的理论预测方法在研究人群中得到证实。由于工作的主要目标是开发一种筛选测定法，以在特定情况下跳过所有样品的测序，因此使用所选SNP进行序列的成对比较。当HVS-1/HVS-2 SNP都用于模拟时，在93.2%的比较中观察到至少两个差异。用个案样本验证该测定法。结果表明，该方法简单，可用于排除目的，节省时间和实验室资源。该测定证实了Salas和Amigo（2010）[1]提出的理论预测。所有的法医优势，如高灵敏度和权力的歧视，以及缺点，如等位基因脱落的发生，在整个文章中进行了讨论。(C)2013爱思唯尔爱尔兰有限公司版权所有。
Mitochondrial DNA (mtDNA) analysis is usually a last resort in routine forensic DNA casework. However, it has become a powerful tool for the analysis of highly degraded samples or samples containing too little or no nuclear DNA, such as old bones and hair shafts. The gold standard methodology still constitutes the direct sequencing of polymerase chain reaction (PCR) products or cloned amplicons from the HVS-1 and HVS-2 (hypervariable segment) control region segments. Identifications using mtDNA are time consuming, expensive and can be very complex, depending on the amount and nature of the material being tested. The main goal of this work is to develop a less labour-intensive and less expensive screening method for mtDNA analysis, in order to aid in the exclusion of non-matching samples and as a presumptive test prior to final confirmatory DNA sequencing. We have selected 14 highly discriminatory single nucleotide polymorphisms (SNPs) based on simulations performed by Salas and Amigo (2010) [1] to be typed using SNaPShot (TM) (Applied Biosystems, Foster City, CA, USA). The assay was validated by typing more than 100 HVS-1/HVS-2 sequenced samples. No differences were observed between the SNP typing and DNA sequencing when results were compared, with the exception of allelic dropouts observed in a few haplotypes. Haplotype diversity simulations were performed using 172 mtDNA sequences representative of the Brazilian population and a score of 0.9794 was obtained when the 14 SNPs were used, showing that the theoretical prediction approach for the selection of highly discriminatory SNPs suggested by Salas and Amigo (2010) [1] was confirmed in the population studied. As the main goal of the work is to develop a screening assay to skip the sequencing of all samples in a particular case, a pair-wise comparison of the sequences was done using the selected SNPs. When both HVS-1/HVS-2 SNPs were used for simulations, at least two differences were observed in 93.2% of the comparisons performed. The assay was validated with casework samples. Results show that the method is straightforward and can be used for exclusionary purposes, saving time and laboratory resources. The assay confirms the theoretic prediction suggested by Salas and Amigo (2010) [1]. All forensic advantages, such as high sensitivity and power of discrimination, as also the disadvantages, such as the occurrence of allele dropouts, are discussed throughout the article. (C) 2013 Elsevier Ireland Ltd. All rights reserved.