Department of Life, Health and Environmental Sciences, University of L'Aquila, L'Aquila, Italy.
Department of Human Studies, University of L'Aquila, L'Aquila, Italy.
PLoS One. 2022 Jun 10;17(6):e0269913. doi: 10.1371/journal.pone.0269913. eCollection 2022.
Molecular biology techniques are increasingly being used in sex identification of skeletal remains when traditional anthropometric analyzes are not successful in identifying sex of remains that are incomplete, fragmented and /or of immature individuals. In the present work, we investigated the possibility of determining sex by using the qPCR-duplex method for both ancient and modern DNA samples. This method involves the co-amplification of two genes in a single reaction system and the subsequent analysis of the fusion curves; the gene sequences used for the construction of suitable primers are those of steroid sulfatase (STS) and testis specific protein Y-linked 1 (TSPY) genes which turned out to be two sensitive markers as they have a detection limit of 60 pg and 20 pg respectively on modern DNA. The validity of the method was verified on modern DNA in which gender was identified in all the samples with 100% accuracy; thus, allowing for the same results as the classic method with amelogenin, but in a faster and more immediate way, as it allows for sex determination solely by analyzing the denaturation curves without having to perform an electrophoretic run. The proposed molecular technique proves to be sensitive and precise even on degraded DNA, in fact on 9 archaeological finds dating from the VII-XII century in which sex had been identified through anthropometric analysis, it confirmed the sex of 8 out of 9 finds correctly.
分子生物学技术在传统人体测量分析无法成功确定不完整、碎片化和/或未成年遗骸的性别时，越来越多地用于骨骼遗骸的性别鉴定。在本工作中，我们研究了使用 qPCR 双重方法确定古代和现代 DNA 样本性别的可能性。该方法涉及在单个反应系统中同时扩增两个基因，并随后分析融合曲线；构建合适引物所使用的基因序列是甾体硫酸酯酶 (STS) 和睾丸特异性蛋白 Y 连锁 1 (TSPY) 基因，事实证明这两个基因作为两个敏感标记，其在现代 DNA 上的检测限分别为 60pg 和 20pg。该方法在现代 DNA 上的有效性得到了验证，其中所有样本的性别均以 100%的准确率进行了识别；因此，与经典的使用 amelogenin 的方法一样，但更快、更直接，因为它仅通过分析变性曲线即可进行性别鉴定，而无需进行电泳运行。所提出的分子技术即使在降解的 DNA 上也具有敏感性和精确性，事实上，在 9 个可追溯到 7-12 世纪的考古发现中，通过人体测量分析确定了性别，它正确地确认了 9 个发现中的 8 个的性别。
