AMERICAN JOURNAL OF PHYSICAL 99:231-238 (1996)

Sex Determination of Ancient Skeletons Using DNA

ANNE C. STONE, GEORGE R. MILNER, SVANTE PAABO, AND MARK STONEKING Department of Anthropology, Pennsylvania State University, University Park, Pennsylvania 16802 (A.C.S.,G.R.M., M.S.); Zoological Institute, university of Munich, 0-80021 Munich, Germany (S.l?) KEY WORDS Ancient DNA, Skeletal Sexing, Amelogenin

ABSTRACT A method for determining the sex of human skeletons was developed using molecular genetic techniques. The amelogenin gene, found on the X and Y chromosomes, was examined using the polymerase chain reaction (PCR) and a nonradioactive dot blot procedure. DNA was analyzed from 20 modern individuals of known sex and 20 skeletons from an archaeolog- ical site in central Illinois dating to A.D. 1300. An independent assessment of the sex of each skeleton was made according to standard osteological methods. The sex of 19 ancient and 20 modern individuals was accurately determined using this molecular genetic technique. Molecular sex determination will be especially useful for juvenile and framentary remains when is diffi- I it cult, -or impossible, to "establish an individual's sex from morphological features. 0 1996 Wiley-Liss, Inc.

Until recently, the size and shape of bones ments that are too large to be routinely ob- were the sole means of establishing the sex tained from ancient specimens. Others rely of skeletons from archaeological or forensic on the presence or absence of a Y-specific contexts. The discovery that DNA can be re- PCR product to indicate sex; for example, covered from old bone (Hagelberg et al., PCR amplification of alphoid repeats found 1989; Horai et al., 1989) provides an oppor- on the Y chromosome (Honda et al., 1990; tunity to determine sex using DNA from the Hummel and Herrmann, 1991, 1993). Al- X and Y chromosomes. Such an analysis is though the presence of a product after PCR particularly useful when standard osteologi- amplification of the alphoid repeats indi- cal methods for determining sex produce, at cates that an individual is male, the absence best, equivocal results, such as when juve- of a product does not necessarily indicate nile or fragmentary remains are examined. that an individual is female. It may simply The ability to establish the sex of such skele- reflect low quantity or quality DNA for am- tal remains contributes important informa- plification or the inhibition of the PCR by tion to forensic cases, and allows the expan- other agents in the sample; both occur fre- sion of archaeological mortuary analyses. quently with ancient DNA (Cooper, 1993; Several molecular methods of sex determi- Hagelberg and Clegg, 1991; Paabo et al., nation using the polymerase chain reaction 1988). (PCR) have been developed for medical or We present a method of determining the forensic purposes (Aasen and Medrano, sex of skeletons using a small fragment (112 1990; Akane et al., 1992; Cui et al., 1994; bp) of the amelogenin gene. Fixed sequence Ebensperger et al., 1989; Handyside et al., differences between the X and Y copies of 1990; Kogan et al., 1987; Nakahori et al., 1991a; Norby and Eriksen, 1992; Witt and Erickson, 1989). Most of these methods, Received February 8, 1995; accepted August 16, 1995 however, are inappropriate for ancient DNA. Address reprint requests to , Department of An- Some require the amplification of DNA frag- thropology, 409 Carpenter Building, University Park, PA 16802.

0 1996 WILEY-LISS, INC. 232 A.C. STONE ET AL. this fragment are then revealed via chemilu- ers for PCR were designed to amplify a small minescent detection with sex-specific oligo- fragment (112 bp) in exon 6 of the amelo- nucleotide probes. This method of sex deter- genin gene. The amelogenin gene, important mination was tested using 20 adult skeletons for enamel development in teeth, is found exhibiting clear male or female morphology on both the X and Y chromosomes outside from a 700-year-old archaeological site. In the pseudoautosomal, or recombining, re- addition, DNA from 10 modern males and 10 gion (Nakahori et al., 1991b). The primers modern females was also tested. Our results amplify corresponding fragments from both demonstrate that this nonradioactive dot the X and Y copies of the amelogenin gene. blot procedure allows rapid, DNA-based sex DNA was extracted from rib bones, usu- determination from ancient as well as mod- ally the 11th or 12th ribs, that were free of ern samples. pathological lesions. These particular speci- mens were chosen for analysis to minimize SAMPLES the destruction of parts of the skeleton fre- quently used.in standard osteological stud- The skeletal sample used for this analysis ies. The outer layer of bone was removed consists of 20 adults from the west-central with a sterile razor blade or a rotary tool Illinois Norris Farms #36 cemetery dating (Sears Craftsman) to prevent contamination to A.D. 1300 (Milner and Smith, 1990). The from previous handling. The bones were cemetery was completely excavated in the then ground to a fine powder using a bone mid-1980s by Illinois State Museum archae- mill (B. Braun Biotech) or an electric coffee ologists, and approximately 260 skeletons grinder (Mr. Coffee). The mill and coffee were found that belong to an Oneota cultural grinder were washed with 1N HCL or bleach component. These bones came from a ceme- and UV irradiated between uses. DNA was tery that is thought to have been used by a extracted from approximately 0.25 g of bone community for a short period of time. They using the silica and guanidine thiocyanate were also exceptionally well-preserved, and extraction protocol described in Hoss and previous research demonstrated that they Paabo (1993). contained sufficient DNA for mitochondria1 The PCR was carried out in a 50 pl volume DNA typing (Stone and Stoneking, 1993). in which a wax-mediated hot start was per- The modern DNA samples were from Indo- formed (Chou et al., 1992). The lower phase nesian individuals of known sex and were of the PCR contained the deoxynucleoside previously extracted from blood (Redd et triphosphates (dNTPs) and primers (Fig. l), al., 1995). while the upper phase contained the DNA, 1 unit of Taq polymerase (Perkin Elmer, METHODS Roche, NJ), and BSA. Both phases contained When choosing the 20 archaeological skel- tris buffer and MgC12.The two phases mixed etons for this analysis, an effort was made as the temperature increased to 94°C during to select specimens that had morphological the first cycle of PCR. After the two phases characteristics that were readily identifiable mixed, the final concentrations were 100 FM as either male or female. In determining the of each dNTP, 20 pmol of each primer, 50 pg sex of these individuals, the greatest weight of bovine serum albumin (fraction V from was placed on several cranial and pelvic fea- Sigma), 67 mM Tris-HCL (pH 8.8), 2.5 mM tures, including the shapes of pubic bones, MgC12, and 5 p1 (approximately 5%) of the the widths of greater sciatic notches, and extracted DNA. Forty cycles of amplification the sizes of preauricular sulci, supraorbital were carried out, with each cycle consisting ridges, mastoid processes, and superior nu- of denaturation at 94°C for 1minute, anneal- chal lines (e.g., Bass, 1971; White, 1991). ing at 65°C for 1 minute, and elongation at Such features were consistent with other 72°C for 1 minute. The PCR products were less precise means of determining the sex of visualized with ethidium bromide in a 2.8% skeletons, including the general robustness NuSieve agarose gel (FMC). For the ancient of bones. samples, PCR product bands were excised For sex determination using DNA, prim- from the gel and placed in 100 p1 of TE or SEX DETERMINATION USING DNA 233

X: CTGCCGCCACAGCCACCTCTGCCTCCGATGTTCCCCATGCAGCCCCTGCCTCCCAT~TTCCT Y: ....T...... A...... C...G...... c.. .. .A...... X : GATCTGACTCTGGAAGCTTGGCCATCAACAGACAAGACCAAGCGGGAGG Y: ...... CA ...... G ...... A.....

PRIMERS : PROBES :

A1 : CTGCTGCC AC AGCC ACC TC TG X : TGCCTCCCATGCTTCCT A2: CCTCCCGCTTGGTCTTGTCTG Y: TGCCCCCCATACTTCCT

Fig. 1. Sequence of the X (nucleotides 2270-2382) and Y (nucleotides 2090-2202) amelogenin gene fragment (Nakahori et al., 1991a) with the location of the primer sequences underlined and the probe sequence in bold. Dots signify identity to the X chromosome copy. Primer and probe sequences are listed below. ddH20.The gel plug was melted at 65°C and (pH 8.011. The denatured DNA was placed 3 pl were used in a 15 cycle reamplification, on the membranes using a manifold (Bio- consisting of denaturation at 94°C for 1min- Rad) and washed with 100 p1 TE. After ute, annealing at 67°C for 1 minute, and briefly blotting the membrane, the DNA was elongation at 72°C for 1 minute. In addition fixed to the membranes with UV irradiation to increasing the amount of DNA present, (550 mJ) in a Stratalinker (Stratagene). the reamplification step avoided using up The membranes were prehybridized in the original PCR product, so that many dot 20-30 ml of 5 X SSPE (0.76 M NaC1, 0.05 blots could be made if necessary. M NaH20P04H20,and 6.25 mM EDTA), 5 X DNA was obtained from a minimum of two Denhardt’s solution (Sambrook et al., 19891, independent extractions per individual. At and 0.5% SDS for 15 minutes at the hybrid- least four separate PCR products were used ization temperature of 45°C. After prehy- for the sex determination of each individual. bridization, 30 p1 of 1 pM X or Y specific To detect contamination by modern DNA, biotinylated probe (Fig. 1) was added and each set of extractions included a negative hybridization was carried out for at least extraction control that contained all extrac- 15 hours. tion reagents except bone powder. In addi- After hybridization, the membranes were tion, PCR negative controls containing all placed in trays and washed twice for 5 min- reagents except for DNA were routinely per- utes at room temperature in a wash solution formed. PCR reagents were also regularly of 2 x SSPE, 0.5% SDS. This was followed tested with primers for mitochondria1 DNA by a wash for 1minute at the hybridization sequences, which are more sensitive to DNA temperature and another wash at room tem- contamination than primers for nuclear sin- perature for 5 minutes. The membranes gle-copy sequences. To further minimize the were then incubated at 42°C in 30 pl of the potential for contamination, all DNA ex- wash solution and 90 pl of streptavidin- tracts and PCRs involving the skeletal sam- horseradish peroxidase (Perkin-Elmer) for ples were prepared in a room dedicated to 10 minutes. Following the incubation step, ancient DNA research that is physically sep- the membranes were washed twice for 1min- arated from the main laboratory. ute each using room temperature wash solu- Dedicated reagents and equipment, includ- tion. They were subsequently placed in 42°C ing gamma-sterilized filter pipet tips (VWR), wash solution and gently agitated for 15 were also used. minutes while the wash solution cooled to Dot blots of the reamplification products room temperature. were prepared using uncharged nylon mem- The membranes were rinsed briefly in 0.1 branes (Biodyne). For each sample, 5 pl of M sodium citrate to remove the remaining PCR product was added to 50 pl of denatur- SDS. Each membrane was agitated for 1 ation solution [0.4 N NaOH, 25 mM EDTA minute in 20 ml of chemiluminescence detec- 234 A.C. STONE ET AL.

Fig. 2. Results of PCR amplification of the 112 bp amelogenin gene fragment using a dilution series of DNA. Lane 1, DNA extracted from a single hair bulb; lanes 2-6, 1 ng, 100 pg, 10 pg, 5 pg, and 1 pg DNA; lane 7, PCR blank. tion reagents prepared according to the man- the Y copy was observed in only five of the ufacturer’s directions (ECL kit, Amersham), ten 5 pg test samples and not in the 1 pg and blotted on 3M Whatman’s paper. While sample. Since one haploid contains the membranes were still damp, they were about 3 pg of DNA, it is likely that PCR is sealed in plastic wrap and placed on X-ray beginning from just one or two copies of the film for 25 minutes. amelogenin gene in these samples. Twenty modern samples from individuals R ESU LTS of known sex were typed blindly; €or these This method for DNA-based sex determi- samples approximately 50 ng of DNA were nation utilizes fixed differences between the used, and the 15 cycle PCR reamplification X and Y copies of the amelogenin gene (Fig. was unnecessary. The sex of 19 of the 20 1).The sensitivity of this method for sex de- modern individuals was correctly ascer- termination of samples containing minute tained, but one male was incorrectly identi- amounts of DNA was established using a fied as female. A second DNA-based sex-typ- dilution series of male DNA (Fig. 2) ranging ing method (Sullivan et al., 1993) also from 1 ng to 1 pg. In addition, ten separate indicated that the individual was female. PCR reactions of both the 5 pg and 1 pg Therefore, this sample appears to have been dilutions were performed. PCR products simply mislabeled in the field and, hence, were obtained from all ten of the reactions the new method correctly identifies this per- containing 5 pg of DNA but only from one son’s sex. of the ten reactions containing 1pg of DNA. The results from a typical PCR amplifica- Dot blot results indicate that the X copy of tion of ancient samples are depicted in Fig. the amelogenin gene fragment was detected 3. DNA from each ancient sample was ex- in all 11 of the amplification products while tracted at least twice, and a minimum of SEX DETERMINATION USING DNA 235

Fig. 3. PCR amplification of the 112 bp amelogenin gene fragment. Lanes 14,bone samples; lanes 5 and 6, extraction blanks; lane 7,PCR blank. The amplification of DNA from the bone samples was successful only in lanes 1 and 2. four PCR products were obtained for dot blot hybridization. A typical dot blot of ancient samples is illustrated in Fig. 4. DNA from the ten ancient females hybridized only with the X specific oligonucleotide, as expected. DNA from nine of the males hybridized with both the X and the Y specific oligonucleotides (Table 1). In several males (such as burial 225 in Figure 4), some amplification prod- ucts hybridized with only one of the two spe- cific probes, consistent with the assumption that the PCR began from one or a very small number of copies. One individual (burial 49) was classified as a male based on pelvic morphology and robust skeletal features, but the PCR prod- ucts hybridized only with the X specific oligo- nucleotide. Although mtDNA was amplified, it was difficult to obtain amplification prod- ucts from the amelogenin gene in this indi- vidual, and it is likely that there is insuffi- cient DNA to determine sex with confidence. Fig. 4. Dot blots probed with X (top) and Y (bottom) An average Of 4.5 PCR amp1ifications was specific oligonucleotides. Al, modern female; A2, mod- required to obtain a single Product for sex ern male; A3,5 pg male control; B1, burial 21 (female); determination in this individual. For the B2, burial 225 (male); B3, burial 19 (male). 236 A.C. STONE ET AL.

TABLE 1. List of burials examined, the sex of each skeleton determined by morphology, results from four PCR products, number of extractions, and number of PCR amplifications required to obtain four independent products PCR 1 PCR 2 PCR 3 PCR 4 Burial Sex X Y X Y X Y X Y Extracts PCRs 19 M X X X X X X 2 4 21 F X X X X 3 9 22 F X X X X 2 5 26 M X X 2 28 F X 3 31 F X 3 35 F X 2 15 36 F X X X X 3 5 38 F X X X X 3 5 47 F X X X X 3 6

49 M X X X X~~ 2 18 50 M X X X X X 2 12 51 F X X X X 4 5 71 M X X X X ND ND 2 14 108 M X X X X X X X 3 9 194 M X X X X 2 11 210 F X X X X 3 5 225 M X 3 23 245 M X 3 17 254 M X 3 4

other ancient samples, an average of 2.4 from intron 1 is amplified that contains a 6- PCR amplifications was sufficient to obtain bp deletion in the X chromosome sequence a product. that is not present in the corresponding Y chromosome sequence. Gill et al. (1994) em- DISCUSSION ployed this technique to determine the sex of The present method to determine sex em- skeletal remains found near Ekaterinburg, ploys PCR and a nonradioactive dot blot pro- Russia, that were reputed to be those of the cedure to examine sequences from the ame- Romanovs, their doctor, and three servants. logenin gene on the X and Y chromosomes. The method described in this paper, like The sex of 19 ancient and 20 modern individ- the technique designed by Sullivan et al. uals was accurately determined giving a suc- (19931, solves the problem encountered cess rate of 95%and 100%respectively. This when using the alphoid Y repeat sequences assumes that one modern sample was misla- for sex determination, because both the X beled, as indicated by this and other sex de- and the Y copies are amplified during PCR. termination methods (Sullivan et al., 1993). Our method, however, appears to be more One ancient individual was classified as a sensitive to very low quantities of DNA, such male based on morphology, but the PCR as those likely to be found in ancient bones. products hybridized only with the X specific A dilution series testing the sensitivity of oligonucleotide. This may be the result of the primers indicates that they can begin insufficient DNA, or mutations in the prim- amplification from as few as one or two cop- ing or oligonucleotide binding sites on the Y ies of the gene (Fig. 2). The Sullivan et al. chromosome copy of the amelogenin gene. (1993) technique, on the other hand, in our Since PCR products for mitochondria1 DNA experience requires at least 100 pg of DNA were also difficult to obtain, it is likely that during 40 cycles of PCR in order to obtain a an insufficient quantity of DNA for sex de- product (data not shown). Attempts to am- termination was present. plify DNA from the bones used in this re- Another molecular technique for sex de- search were not successful using the Sulli- termination that has been applied to skeletal van et al. (1993) primers (data not shown). remains was developed by Sullivan et al. Because of damage and degradation, ana- (1993). For this method, which also uses the lyzing ancient DNA is difficult, and it re- amelogenin gene, a short DNA fragment quires great care to prevent contamination SEX DETERMINATION USING DNA 237 from modern sources. In addition to taking pology Department of the Pennsylvania the precautions that were mentioned above, State University and by a NSF dissertation all results must be confirmed with indepen- improvement grant 9408398 to AS. This re- dent extracts. For sex determination, sam- search was initiated while A.S. was a Ful- ples that do not hybridize with the Y specific bright Scholar in the laboratory of S.P. We oligonucleotide should be examined using at thank 0.Handt and M. Hoss for helpful dis- least four separate PCR products in order to cussion. have a 94% certainty that no Y copies are present. This is especially true if the amount of nuclear DNA present is low (as indicated LITERATURE CITED by difficulty in amplifying bands), possibly Aasen E, and Medrano JF (1990) Amplification of the indicating that the PCR begins from only one ZFY and ZFX genes for sex identification in , copy (Fig. 4 and Table 1). In such instances, cattle, sheep and goats. BioTechnology 8t1279-1281. 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Japan Acad. 65(B):229-233. funded by a Hill Fellowship from the Anthro- Hoss M, and Paabo S (1993) DNA extraction from Pleis- 238 A.C. STONE ET AL.

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