Prediction of autosomal STR typing success in ancient and Second World War bone samples.
Prediction of autosomal STR typing success in ancient and Second World War bone samples.
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DOI：
10.1016/j.fsigen.2016.11.004
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发表时间：
2017-03
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通讯作者：
I. Zupanič Pajnič;T. Zupanc;J. Balažič;Živa Miriam Geršak;O. Stojković;Ivan Skadrić;Matija Črešnar
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作者：
I. Zupanič Pajnič;T. Zupanc;J. Balažič;Živa Miriam Geršak;O. Stojković;Ivan Skadrić;Matija Črešnar
人类特异性定量聚合酶链式反应(QPCR)是近10年来发展起来的一种用于法医鉴定的DNA定量技术，它具有准确、灵敏、客观、时效和自动化程度高等优点，是首选的DNA定量技术。使用商用定量试剂盒从一次定量反应中收集到的信息量已经从核DNA的数量增加到男性DNA的数量、抑制剂的存在以及最近的DNA降解程度。在灾难受害者、失踪人员和战争冲突受害者的骨骼遗骸样本中，DNA通常被降解。因此，能够评估降解程度的新的商用qPCR试剂盒有可能预测下游短串联重复序列(STR)分型的成功。这项研究的目的是验证使用PowerQuant试剂盒的量化步骤是否适合作为对古代和第二次世界大战(WWII)骨骼遗骸进行常染色体STR分型成功的筛查方法。我们分析了从五个考古遗址和四个来自斯洛文尼亚的二战乱葬坑出土的60具骨架。骨头被清理干净，表面污染被去除，骨头被磨成粉末。从0.5 mg骨粉中提取基因组DNA。DNA是用Biorobot EZ1设备提纯的。在PowerQuant定量后，DNA样本使用NGM试剂盒进行常染色体STR扩增。提取的DNA含量高达2.51 ng/g粉末。在所分析的任何骨骼中都没有检测到抑制作用。82%的二战骨骼给出了完整的剖面，而73%的古代骨骼给出了不适合解释的剖面。四种骨提取液没有产生可检测到的扩增结果或量化结果为零，并且没有从它们中获得任何图谱。只有在检测到短常染色体(Auto)和长降解(Deg)PowerQuant靶点的骨提取液中才能产生完整或有用的部分轮廓。结论：仅当同时检测到Auto和Deg靶时，才应对用PowerQuant量化后的陈旧骨进行STR分型，而不考虑[Auto]/[Deg]比率。根据Deg片段扩增的成功与否可以预测STR分型成功与否。PowerQuant试剂盒能够识别不会使用NGM试剂盒产生有用STR图谱的骨DNA样本，它可以作为从古代和二战骨骼遗骸中获得的骨提取物的常染色体STR分型成功的预测指标。
Human-specific quantitative PCR (qPCR) has been developed for forensic use in the last 10 years and is the preferred DNA quantification technique since it is very accurate, sensitive, objective, time-effective and automatable. The amount of information that can be gleaned from a single quantification reaction using commercially available quantification kits has increased from the quantity of nuclear DNA to the amount of male DNA, presence of inhibitors and, most recently, to the degree of DNA degradation. In skeletal remains samples from disaster victims, missing persons and war conflict victims, the DNA is usually degraded. Therefore the new commercial qPCR kits able to assess the degree of degradation are potentially able to predict the success of downstream short tandem repeat (STR) typing. The goal of this study was to verify the quantification step using the PowerQuant kit with regard to its suitability as a screening method for autosomal STR typing success on ancient and Second World War (WWII) skeletal remains. We analysed 60 skeletons excavated from five archaeological sites and four WWII mass graves from Slovenia. The bones were cleaned, surface contamination was removed and the bones ground to a powder. Genomic DNA was obtained from 0.5 g of bone powder after total demineralization. The DNA was purified using a Biorobot EZ1 device. Following PowerQuant quantification, DNA samples were subjected to autosomal STR amplification using the NGM kit. Up to 2.51 ng DNA/g of powder were extracted. No inhibition was detected in any of bones analysed. 82% of the WWII bones gave full profiles while 73% of the ancient bones gave profiles not suitable for interpretation. Four bone extracts yielded no detectable amplification or zero quantification results and no profiles were obtained from any of them. Full or useful partial profiles were produced only from bone extracts where short autosomal (Auto) and long degradation (Deg) PowerQuant targets were detected. It is concluded that STR typing of old bones after quantification with the PowerQuant should be performed only when both Auto and Deg targets are detected simultaneously with no respect to [Auto]/[Deg] ratio. Prediction of STR typing success could be made according to successful amplification of Deg fragment. The PowerQuant kit is capable of identifying bone DNA samples that will not yield useful STR profiles using the NGM kit, and it can be used as a predictor of autosomal STR typing success of bone extracts obtained from ancient and WWII skeletal remains.