Use of a Multiplex Polymerase Chain Reaction Assay in the Sex Typing of DNA Extracted from Archaeological Bone

We developed an original method that allows the simultaneous molecular amplification and analysis of human chromosome X- and Y-specific sequences from cortical compact bone using the polymerase chain reaction (PCR). The strategy is based on sequential amplification using external and internal primers. The targeted loci are SRY for chromosome Y and DXZ4 for chromosome X. Internal and external primer pairs for both loci were selected in order to allow the simultaneous amplification of short sequences of distinct molecular weight in the same reaction. Ethidium bromide stained PCR products were analysed on 2 per cent agarose gels. The presence of two distinct amplification bands permitted the fast and unequivocal identification of male bones, whereas female bones were identified by the presence of a single band. The technique was validated by comparative analysis of modern human DNAs of known sex and of DNAs from medieval bone samples whose sex could be determined using conventional anthropological methods. We then analysed bone samples from 40 burials found under the Church of St Maria Aprutiensis in Teramo, Abruzzo. These burials were dated to AD 800-1200 by ¹⁴C accelerated mass spectrometry radiometric analysis. Molecular analysis allowed the unambiguous identification of sex in all the samples studied. These results indicate that molecular techniques represent useful and often critical additions to anthropological studies of ancient human remains.