骨骼和牙齿代表了古代DNA研究和法医案例中的一个共同发现,即使经过长时间的埋葬。基因分型是个人鉴定骨骼遗骸的金标准,但是,成功进行此类样品的DNA分型有两个主要因素:(1)建立有效的DNA提取方法;(2)鉴定最适合下游遗传分析的骨骼元素。在本文中,基于使用Na2EDTA处理0.5g脱钙的骨粉的方案被证明适用于使用Maxwell®FSCDNAIQ™CasewayKit(Promega,麦迪逊,WI,美国)。将该方法在DNA回收和质量方面的性能与基于Qiagen技术和试剂盒的完全去矿化提取方案进行比较。根据DNA恢复和DNA降解指数(p值≥0.176;r≥0.907)评分无统计学意义。这种新的DNA提取方案应用于88个骨骼样本(41个股骨,19岩骨,12颗掌骨和16颗磨牙)据称属于在克雷斯岛(克罗地亚)的一个万人坑中发现的27名第二次世界大战意大利士兵。Quantifiler人类DNA定量试剂盒进行的qPCR结果显示,所有岩骨的定量值都高于最低限度(lLOQ;23pg/µL),而其他骨骼类型显示,在大多数情况下,更低的DNA。复制STR-CE分析显示成功键入(即,>12个标记)在岩骨上的所有测试中,其次是掌骨(83.3%),股骨(52.2%)和牙齿(20.0%)。全谱(22/22常染色体标记)主要在岩骨(84.2%),其次是掌骨(41.7%)。随机放大伪像,如脱落或脱落,在岩骨中发生的频率为1.9%,而当从其他骨元素中回收的DNA被扩增时,它们更高(股骨中高达13.9%)。总的来说,这项研究的结果证实,岩骨在回收的DNA的数量和质量方面优于其他骨骼元素;因此,如果可用,它应该始终是基因检测的首选。此外,我们的结果强调了准确规划DVI操作的必要性,这应该由一个多学科的团队来执行,以及确定其他合适的骨骼元素进行基因检测的棘手问题。总的来说,本文提出的结果支持需要采用与老年骨骼遗骸成功基因检测呈正相关的分析前策略,以降低成本和分析时间。
Bones and teeth represent a common finding in ancient DNA studies and in forensic casework, even after a long burial. Genetic typing is the gold standard for the personal identification of skeletal remains, but there are two main factors involved in the successful DNA typing of such samples: (1) the set-up of an efficient DNA extraction method; (2) the identification of the most suitable skeletal element for the downstream genetic analyses. In this paper, a protocol based on the processing of 0.5 g of bone powder decalcified using Na2EDTA proved to be suitable for a semi-automated DNA extraction workflow using the Maxwell® FSC DNA IQ™ Casework Kit (Promega, Madison, WI, USA). The performance of this method in terms of DNA recovery and quality was compared with a full demineralisation extraction protocol based on Qiagen technology and kits. No statistically significant differences were scored according to the DNA recovery and DNA degradation index (p-values ≥ 0.176; r ≥ 0.907). This new DNA extraction protocol was applied to 88 bone samples (41 femurs, 19 petrous bones, 12 metacarpals and 16 molars) allegedly belonging to 27 World War II Italian soldiers found in a mass grave on the isle of Cres (Croatia). The results of the qPCR performed by the Quantifiler Human DNA Quantification kit showed values above the lowest Limit of Quantification (lLOQ; 23 pg/µL) for all petrous bones, whereas other bone types showed, in most cases, lower amounts of DNA. Replicate STR-CE analyses showed successful typing (that is, >12 markers) in all tests on the petrous bones, followed by the metacarpals (83.3%), femurs (52.2%) and teeth (20.0%). Full profiles (22/22 autosomal markers) were achieved mainly in the petrous bones (84.2%), followed by the metacarpals (41.7%). Stochastic amplification artefacts such as drop-outs or drop-ins occurred with a frequency of 1.9% in the petrous bones, whereas they were higher when the DNA recovered from other bone elements was amplified (up to 13.9% in the femurs). Overall, the results of this study confirm that petrous bone outperforms other bone elements in terms of the quantity and quality of the recovered DNA; for this reason, if available, it should always be preferred for genetic testing. In addition, our results highlight the need for accurate planning of the DVI operation, which should be carried out by a multi-disciplinary team, and the tricky issue of identifying other suitable skeletal elements for genetic testing. Overall, the results presented in this paper support the need to adopt preanalytical strategies positively related to the successful genetic testing of aged skeletal remains in order to reduce costs and the time of analysis.