Madel Maria-Bernadette, Niederstätter Harald, Parson Walther
Institute of Legal Medicine, Medical University of Innsbruck, Innsbruck, Austria.
Institute of Legal Medicine, Medical University of Innsbruck, Innsbruck, Austria; Forensic Science Program, The Pennsylvania State University, University Park, PA, USA.
Forensic Sci Int Genet. 2016 Nov;25:166-174. doi: 10.1016/j.fsigen.2016.09.001. Epub 2016 Sep 3.
Sexing of biological evidence is an important aspect in forensic investigations. A routinely used molecular-genetic approach to this endeavour is the amelogenin sex test, which is integrated in most commercially available polymerase chain reaction (PCR) kits for human identification. However, this assay is not entirely effective in respect to highly degraded DNA samples. This study presents a homogeneous PCR assay for robust sex diagnosis, especially for the analysis of severely fragmented DNA. The introduced triplex for the X and Y chromosome (TriXY) is based on real-time PCR amplification of short intergenic sequences (<50bp) on both gonosomes. Subsequent PCR product examination and molecular-genetic sex-assignment rely on high-resolution melting (HRM) curve analysis. TriXY was optimized using commercially available multi-donor human DNA preparations of either male or female origin and successfully evaluated on challenging samples, including 46 ancient DNA specimens from archaeological excavations and a total of 16 DNA samples extracted from different segments of eight hair shafts of male and female donors. Additionally, sensitivity and cross-species amplification were examined to further test the assay's utility in forensic investigations. TriXY's closed-tube format avoids post-PCR sample manipulations and, therefore, distinctly reduces the risk of PCR product carry-over contamination and sample mix-up, while reducing labour and financial expenses at the same time. The method is sensitive down to the DNA content of approximately two diploid cells and has proven highly useful on severely fragmented and low quantity ancient DNA samples. Furthermore, it even allowed for sexing of proximal hair shafts with very good results. In summary, TriXY facilitates highly sensitive, rapid, and costeffective genetic sex-determination. It outperforms existing sexing methods both in terms of sensitivity and minimum required template molecule lengths. Therefore, we feel confident that TriXY will prove to be a reliable addition to the toolbox currently used for sex-typing in forensic genetics and other fields of research.
生物证据的性别鉴定是法医调查中的一个重要方面。在这项工作中，一种常规使用的分子遗传学方法是牙釉蛋白性别测试，它被整合到大多数市售的用于人类身份识别的聚合酶链反应（PCR）试剂盒中。然而，对于高度降解的DNA样本，该检测方法并不完全有效。本研究提出了一种用于可靠性别诊断的均相PCR检测方法，特别是用于分析严重片段化的DNA。引入的X和Y染色体三重检测法（TriXY）基于对两条性染色体上短基因间序列（<50bp）的实时PCR扩增。随后的PCR产物检测和分子遗传学性别判定依赖于高分辨率熔解（HRM）曲线分析。TriXY使用市售的男性或女性来源的多供体人类DNA制剂进行了优化，并在具有挑战性的样本上成功进行了评估，包括46个来自考古发掘的古代DNA样本，以及从男性和女性供体的8根毛发轴不同片段中提取的总共16个DNA样本。此外，还检测了灵敏度和跨物种扩增，以进一步测试该检测方法在法医调查中的实用性。TriXY的闭管形式避免了PCR后样本处理，因此，显著降低了PCR产物携带污染和样本混淆的风险，同时减少了劳动力和财务费用。该方法对大约两个二倍体细胞的DNA含量敏感，并且已被证明对严重片段化和低含量的古代DNA样本非常有用。此外，它甚至能够对近端毛发轴进行性别鉴定，结果非常好。总之，TriXY有助于进行高度灵敏、快速且经济高效的基因性别判定。在灵敏度和所需最小模板分子长度方面，它都优于现有的性别鉴定方法。因此，我们相信TriXY将被证明是目前用于法医遗传学和其他研究领域性别分型的工具库中一个可靠的补充。
