• Z Lin, T Kondo, T Minamino, M Ohtsuji, J Nishigami, T Takayasu, R Sun, and T Ohshima.
• Department of Legal Medicine, Kanazawa University School of Medicine, Japan.
• Forensic Sci. Int. 1995 Oct 30;75(2-3):197-205.

AbstractSex determination was performed by the polymerase chain reaction (PCR) on eight adult mummies and one child mummy which were discovered at Taklamakan desert in 1912 and now belong to the Lüshun Museum in China. Archaeologically, these mummies were humans living in the seventh century, that is, more than 1300 years ago. Putative sex determination was performed based on external morphology for six of the eight adults, but it was impossible for the other two adults and one child mummy due to marked destruction on the external morphology. Hair, muscle and skin samples were then collected from each adult mummy, and skin and rib samples from the child mummy. Forty PCR cycles were performed as follows: denaturation at 94 degrees C for 40 s, annealing at 55 degrees C for 30 s and extension at 72 degrees C for 1 min. The primer and PCR reaction mixture were prepared according to the report by Witt and Erickson (M. Witt and R. P. Erikson, A rapid method for detection of Y-chromosomal DNA from dried blood specimens by the polymerase chain reaction. Hum. Genet., 82 (1989) 271-274)). Two different pairs of primer were used. One was X1, X2 (X1: 5'-AATCATCAAATGGAGATTTG-3'; X2: 5'-GTTCAGCTCTGTGAGTGAAA-3') to flanking the 170 bp fragment of the alphoid repeats on the human X chromosome, and the other was Y11, Y22 (Y11: 5'-ATGATAGAAACGGAAATATG-3'; Y22: 5'-AGTAGAATGCAAAGGGCTC-3') to flanking the 130 bp fragment of the alphoid repeats on the human Y chromosome. Extracted DNA solutions from mummy samples was purified using a spin column (T. Yoshii, K. Tamura, T. Taniguchi, K. Akiyama and I. Ishiyama, Water-soluble eumelanin as a PCR-inhibitor and a simple method for its removal. Jpn. J. Legal Med., 47 (1993) 323-329 (in Japanese with English abstract) for removing PCR-inhibitors, and bovine serum albumin (BSA) was employed to inhibit the remaining impurities even after the purification with the column. In six adult cases where the putative sex was determined from external morphology, the sex in five cases was consistent with that by PCR using hair, muscle, and skin samples, but the other one was inconsistent. In two adult cases where sex estimation was externally impossible, the sex was determined to be male because both X-specific and Y-specific bands were clearly detected. The child mummy was definitely male. This study shows that the sex determination was possible by the PCR method even with very ancient human samples > 1300 years old, that spin column was useful for removing impurities in the DNA solution from ancient human samples and that the BSA of optimum concentration suppressed the action of the PCR-inhibitory factors.

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