A qPCR-duplex assay for sex determination in ancient DNA

Abstract

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.

Estimation of DNA Degradation in Archaeological Human Remains

“…The excavation and recovery of the archaeological remains were carried out following a protocol to prevent possible contamination of the bone remains with modern DNA [ 33 ]. The samples selected for DNA extraction were treated as in previous works [ 7 , 34 ]. The bone remains come from three intact graves (S.46, S.48, and S.50) and from two reworked but well-preserved graves (S.44 and S.45).…”

“…The experiments were conducted in two separate laboratories, one pre-PCR and one post-PCR, equipped with dedicated equipment [ 6 , 7 ]. The bone powder samples have been prepared in the pre-PCR laboratory by milling a small area of bone findings [ 34 ]. The DNA was extracted from the samples through the Geneall ® Exgene TM Genomic DNA micro ® kit (Seoul, Republic of Korea) following the manufacturer’s instructions.…”

“…The reaction mix (20 μL total volume) contained: 10 μL PowerUp SYBR Green Master Mix (2U×) (Applied Biosystems), 2 μL mix of specific forward and reverse primer (5 μM), DNA at various concentrations, and variable ddH 2 O up to 20 µL. Amplification conditions are as in previous works [ 34 ]. For each reaction, the efficiency (>90%), linearity, dynamic range, sensitivity, and specificity were calculated by studying the melting profiles of the amplicons.…”

Peptide analysis of tooth enamel – A sex estimation tool for archaeological, anthropological, or forensic research

“…Molecular biology and genetics can use aDNA at the individual level to identify the biological sex of a skeleton, make phenotypic inferences from an individual's genotype, and identify specific pathogens within an infected individual [27]. Molecular biology techniques are increasingly used to identify the sex of skeletal remains when traditional anthropometric analyses do not successfully identify the sex of remains that are incomplete, fragmented, and/or refer to immature individuals [28][29][30]. As Raff [27] reports, aDNA research cannot be done in regular molecular biology laboratories.…”