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.

Fig 1. Agarose gel electrophoresis of amplicons on modern DNA.

Electrophoretic analysis of amplicons generated in qPCR using the primers TSPY67, TSPY119, STS89, STS95, STS158Y, STS154/116, with male (♂) and female (♀) human DNA. M) SharpMass ™ 50 molecular weight marker—Ready-to-load DNA Ladder (Euroclone S.p.A). C) control without template DNA. Agarose gels are 2.2% and stained with ethidium bromide as reported in Materials and Methods.

Fig 2. Agarose gel electrophoresis of amplicons produced in qPCR with STS154/116 and AMEL106/112 primers on modern DNA.

(A) short electrophoretic run: in lanes 1 and 2 amplicons obtained with AMEL106/112 on female and male DNA respectively; in lanes 3 and 4 amplicons obtained with STS154/116 on female DNA; in lane 5 amplicons obtained with STS154/116 on male DNA. (B) long electrophoretic run: in lanes 1 and 2 amplicons obtained with AMEL106/112 on female and male DNA respectively; in lanes 3 and 4 amplicons obtained with STS154/116 on female DNA; in lane 5 amplicons obtained with STS154/116 on male DNA. M) SharpMass ™ 50 molecular weight marker—Ready-to-load DNA Ladder (Euroclone S.p.A). Agarose gels are 2.9% and stained with ethidium bromide as reported in Materials and Methods.

Fig 3. Thermal denaturation curves of amplification products in qPCR-duplex on modern DNA.

Amplification products were obtained using 400 pg male (♂) and female (♀) DNA as a template in a 20 μl reaction system. The pairs of primers used were: STS158Y/STS89 showing two amplification products, having Tm of 72.2°C and 76.5°C, and a single amplification product, having a Tm of 72.4°C, using male and female DNA respectively; STS89/TSPY119 showing two amplification products, having Tm of 72.7°C and 81.5°C, and a single amplification product, having a Tm of 72.7°C, using male and female DNA respectively; STS95/TSPY67 showing two amplification products, having Tm of 73.5 and 79.2°C, and a single amplification product, having a Tm of 72.7°C, using male and female DNA respectively.

Fig 4. Agarose gel electrophoresis of the amplification products in qPCR-duplex on modern DNA.

The three gels were loaded respectively with the qPCR-duplex reaction products obtained with the pairs of primers STS89/TSPY119, STS95/TSPY67 and STS158Y/STS89: (C) control without DNA; lane 2 male DNA; lane 3 female DNA. M) SharpMass ™ 50 molecular weight marker—Ready-to-load DNA Ladder (Euroclone S.p.A). The gels were made with 2.2% agarose and stained with 0.5 μg / mL ethidium bromide.

Fig 5. Thermal denaturation curves of the amplification products in qPCR-duplex on ancient DNA.

Each extracted ancient DNA sample 150 pg was amplified in duplicate in qPCR with the pairs of primers STS158/STS89 (A), STS89/TSPY119 (B) and STS95/TSPY67 (C). In B and C the male samples (S42, S43, S45, S46, S48) show two peaks while the female samples (S44, S47, S49, S50) only one peak. In A all samples show only one peak.

Fig 6. Agarose gel electrophoresis of the amplification products on ancient DNA.

(A) The gels were loaded with the qPCR-duplex reaction products obtained with the pairs of primers STS89/TSPY119, STS95/TSPY67 on male (S42, S43, S45, S46, S48) and female (S44, S47, S49, S50) DNA. In the channels (1,3,5,7,9,11,13,15,17), the products of qPCR with the pair of primers STS89/TSPY119 are shown. In the channels (2,4,6,8,10,12,14,16) are reported the products of qPCR with the pair of primers STS95/TSPY67. The gels were made with 2.2% agarose and stained with 0.5 μg/mL ethidium bromide. (B) Amplification fragments generated in qPCR on ancient DNA using the primers AMEL106/112. The gels were made with 2.9% agarose and stained with 0.5 μg/mL ethidium bromide. M) marker of molecular weight, Sharp Mass ™ 50—Ready-to-load DNA Ladder (Euroclone S.p.A).

Fig 7. Sensitivity analysis in qPCR.

(A) the amplification graphs show the fluorescence variation (Delta Rn) with respect to the number of cycles for the different amplicons obtained with the primers: ALU127, TSPY119, TSPY67, STS89, STS95, STS158Y; 400 pg of male and female DNA were used in the reaction system. B) the graph shows the mean Ct values of genes amplified using DNA from both sexes. The blue columns represent the male DNA, the red columns the female DNA.