Brief communication Multiplex XY-PCR improves sex identification in aDNA analysis.код для вставкиСкачать
AMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY 121:337–341 (2003) Brief Communication: Multiplex X/Y-PCR Improves Sex Identiﬁcation in aDNA Analysis Diane Schmidt,* Susanne Hummel, and Bernd Herrmann Institute for Zoology and Anthropology, Historical Anthropology and Human Ecology, University Göttingen, 37073 Göttingen, Germany KEY WORDS PCR; sex identiﬁcation; ancient DNA; degraded DNA; X-chromosome; Y-chromosome; STR ABSTRACT This study introduces a polymerase chain reaction (PCR)-based multiplex approach to improve the certainty of molecular sex identiﬁcation on archaeological skeletal material. We coampliﬁed amelogenin, two X-chromosomal short tandem repeats (STRs) (DXS6789 and DXS9898), and two Y-speciﬁc STRs (DYS391 and DYS392). The ampliﬁcation results of this multiplex approach back each other up, and enable a reliable sex identiﬁcation. This coampliﬁcation of X- and Y-speciﬁc markers in a multiplex assay combines the added advantage of positive identiﬁcation of both female and male individuals with raising the validity of the diagnosis by obtaining multiple data simultaneously. This multiplex system was successfully applied to 3,000-year-old bone material. Am J Phys Anthropol 121: 337–341, 2003. © 2003 Wiley-Liss, Inc. One of the questions ﬁrst asked of excavated skeletal remains is its sex. Sex identiﬁcation is one of the most basic requirements for any reconstruction of demographic and social structure in human populations. Morphological and morphometrical methods (Herrmann et al., 1990) give reliable and consistent results for adult individuals. However, for determining the sex of children, these methods are less reliable (Schutkowski, 1993). The development of molecular methods, such as the polymerase chain reaction (PCR), for analyzing the remains of children as well as of adults for whom only a partial skeleton remains, has led to signiﬁcant improvements in sex determination. PCRbased sexing is a valuable tool for obtaining data in archaeological studies on remains of infants, as was demonstrated in the case of infanticide victims of a Roman brothel where a bias toward males was found (Faerman et al., 1997). Moreover, molecular sex determination can also play an important role in examining hypotheses based on data obtained with other methods. An example is the investigation of the early modern burial site Aegerten (Switzerland). There, subjects of the study were stillborn and neonate individuals buried along the walls of the church. Morphometrical sex determination showed a clear female bias (Bacher et al., 1990). Therefore, the question arose as to whether there was biased mistreatment by the society towards female neonates. This hypothesis was rejected after PCR-based sex determination. The results of molecular analysis provided evidence for an equivalent balance of both sexes (Lassen et al., 1997, 2000), and even revealed a disproportion of male individuals that died during the last trimester of pregnancy, similar to observations found in modern populations (e.g., Bartels et al., 1990). For the molecular sex determination performed in those studies, the amelogenin gene (Nakahori et al., 1991; Mannucci et al., 1994; Bailey et al., 1992; Lassen et al., 1995; Stone et al., 1996) was ampliﬁed, exhibiting a length dimorphism for the Xand Y-chromosome (106 and 112 bp). Here, the advantage over earlier attempts to determine sex from archaeological samples focusing on Y-speciﬁc markers (e.g., Hummel and Herrmann, 1991; Honda et al., 2001) was in the simultaneous detection of both an X- and Y-chromosomal sequence. Still, allelic dropouts due to degradation of the DNA were found to occur, potentially leading to incorrect determination of male individuals as females in cases of the Y-chromosomal sequence failing to amplify. The only way to decrease the rate of such false female determinations was by increasing the number of ampliﬁcations. In this study, we also describe the use of molecular methods to determine the sex of archaeological © 2003 WILEY-LISS, INC. Grant sponsor: Bundesministerium für Bildung und Forschung. *Correspondence to: Diane Schmidt, Institute for Zoology and Anthropology, Historical Anthropology and Human Ecology, University Göttingen, Bürgerstrasse 50, 37073 Göttingen, Germany. E-mail: email@example.com Received 14 March 2002; accepted 15 August 2002. DOI 10.1002/ajpa.10172 338 D. SCHMIDT ET AL. TABLE 1. Primers used in this study: sequences, dye labelling, and allele ranges Primer Sequence (5⬘ to 3⬘) Dye label Repeats (alleles) Size of alleles (bp) DXS6789, upper DXS6789, lower DXS9898, upper DXS9898, lower Amelogenin, upper Amelogenin, lower DYS391, upper DYS391, lower DYS392, upper DYS392, lower GTTGGTACTTAATAAACCCTCTTTT GGATCCCTAGAGGGACAGAA CACACCTACAAAAGCTGAGATATAT CATCCAGATAGACAGATCAATAGATT CCCTGGGCTCTGTAAAGAATAGT ATCAGAGCTTACACTGGGAAGCTG TTGTGTATCTATTCATTCAATCATA GGAATAAAATCTCCCTGG T CAAGAAGGAAAACAAATTTTTT GGATCATTAAACCTACCAATC NED 14–25 120–164 HEX 8.3–15 130–155 HEX X–Y 106–112 7–14 138–166 6–16 91–121 6FAM 6FAM ampliﬁed ranged from 91–166 bp due to the new primer design we conducted. MATERIALS AND METHODS Initially, the multiplex PCR system was tested on modern saliva samples. Then it was applied to 3,000-year-old skeletal material from the Lichtenstein cave (a Bronze Age cave in the Harz Mountains) where extensive genetic analysis has already been performed (Schultes, 2000), including a molecular sex determination through the analysis of the amelogenin gene as a part of the AmpFlSTR Proﬁler Plus™ kit (ABI). Extraction Fig. 1. Agarose gel, showing overall ampliﬁcation success for X-/Y-multiplex approach for historical samples from the Lichtenstein cave (DO). Ampliﬁcation products ranged from 91–166 bp (partially overlapping in size), below 75-bp primer dimers. MW, 1 kb (kilobase) molecular weight standard; bp, base pairs. skeletal remains. However, our study focused on improving the certainty of sexing by reducing false interpretations due to allelic dropout in the amelogenin system or those rare cases of a deletion of its Y-chromosomal part (Santos et al., 1998). We aimed to positively identify both females and males by simultaneously analyzing several gonosomal markers. For that purpose, a multiplex PCR was used to amplify the amelogenin gene together with two X- and two Y-speciﬁc short tandem repeats (STRs). This strategy, of amplifying multiple X- and Y-chromosomal sequences, has been used successfully in forensics (Pﬁtzinger et al., 1993; Tun et al., 1999; Young et al., 2000; Honda et al., 2001), and is recommended for archaeological investigations (Brown, 1998). The multiplex PCR approach presented here is particularly suitable for application to highly degraded DNA, as the lengths of the products Saliva samples were extracted with Chelex 100™, basically following the protocol described by Walsh et al. (1991). The extraction of DNA from archaeological bone samples was performed using a phenol/ chloroform-based method (for details, see Baron et al., 1996; Lassen et al., 1996). All precautions and steps of sample preparation and extraction followed laboratory routines for ancient DNA (aDNA) handling (Schmidt et al., 1995; Lassen et al., 1996; Gerstenberger et al., 1998). PCR amplification for sex determination Amelogenin was ampliﬁed revealing a sex-speciﬁc length dimorphism. For the detection of the Y-chromosome, two polymorphic Y-speciﬁc markers (DYS391 and DYS392; Kayser et al., 1997) were ampliﬁed.1 For the detection of the X-chromosome, the markers DXS6789 (Hering et al., 2001), DXS9898 (Hering and Szibor, 2000) were used, which are also polymorphic STRs. Males show only one allele for each STR system. Females exhibit two alleles, and due to the comparatively high heterozygosity (H) of the markers (H ⫽ 0.78 for DXS6789 and 0.75 for DXS9898), one might expect at least one of the systems to be heterozygous in most individuals. DXS6789 is located on the short arm of X-chromosome Xq22.3 (Hering et al., 2001). DXS9898 is found at the Xq21.33 pericentric region (Hering and Szi- 1 The primer sequences used in this study are part of the diploma thesis of Boris Mueller (Institute for Zoology and Anthropology, Historical Anthropology and Human Ecology, University Göttingen). X/Y MULTIPLEX PCR FOR SEX IDENTIFICATION 339 Fig. 2. Electropherogram, showing ampliﬁcation results for a male (DO 1911) and female (DO 3742) individual from the Lichtenstein cave. Numbers (100, 139,150, 160, 200) on top of small peaks indicate size (in bp) of internal lane standard GS 500. In male individual (top), in addition to amelogenin, the two Y-STRs (DYS391 and DYS392) ensure sex identiﬁcation. In the female (bottom), DO 3742, Y-chromosomal signals are completely absent, while both X loci (DXS6789 and DXS9898) are expressed as heterozygous, which represents positive proof for female individuals. bor, 2000). Thus, as degradation of DNA starts at the telomeric ends of the chromosome (Hummel et al., 1999), the possibility is assumed to be high for amplifying both markers, even from severely degraded DNA. All ampliﬁcations were carried out from at least two independent extracts of each individual. Primer sequences and allele lengths are given in Table 1. Amplification parameters F1–5 l DNA extract was added to a mixture containing KCl (50 mM), 10 mM Tris-HCl (GeneAmp 10 ⫻ PCR Buffer II, Applied Biosystems), 2 mM MgCl2, 2.5 U AmpliTaq Gold™, 200 M dNTPs, 0.4 M primer DXS6789, 0.2 M primer DXS9898, 0.35 M primer DYS391, 0.2 M primer DYS392, 0.2 M primer amelogenin. Cycling parameters Eleven min initial activation was followed by 50 –55 cycles of 40 sec denaturation at 94°C, 1 min hybridization at 50°C and 1 min extension at 72°C, followed by a ﬁnal extension for 30 min at 60°C. Detection Overall ampliﬁcation success was determined by (2.5– 4%) agarose gel electrophoresis. The speciﬁc detection and determination of the alleles were performed with ABI 373A stretch (Applied Biosystems), employing the fragment length detection software package 672 GENESCAN™ Analysis (Applied Biosystems). RESULTS AND DISCUSSION The initial investigation to test the primers on recent samples was successful (data not shown). Ampliﬁcation of the historical samples also showed good ampliﬁcation results for all individuals (Figs. 1, 2). The sexing of historical samples was in full agreement with the earlier data (Schultes, 2000) our study had reference to. Allele determinations for 11 individuals (7 females and 4 males) are shown in Table 2. In our study, all female individuals were heterozygous for at least one X-STR. In combination with the absence of Y-chromosomal ampliﬁcation results, this supported the validity of the amelogenin results and ensured a positive sex determination as female. This advantage of the coampliﬁed X- and Y-STRs conﬁrming each other can be demonstrated in the case of an ambiguous result for amelogenin in a single PCR reaction of DO 3742 (female). There, in addition to the clear signal for the X-allele, a weak signal of the size of its Y-allele was detected. Since there were simultaneously ampliﬁed heterozygous results for both X-STRs and no Y-STR signals at all, even for this single PCR, the sexing as female was sure, in spite of the appearance of this (not reproducible) artifact. The combined ampliﬁcation of X- and Y-speciﬁc markers enables ancient DNA investigators to reliably determine the biological sex even of skeletal 340 D. SCHMIDT ET AL. TABLE 2. Allele determination from bronze age bone samples1 Sample/ref. sex from earlier study DO DO DO DO DO DO DO DO DO DO DO Amelogenin DXS6789 DXS9898 DYS 392 DYS 391 X/X X/Y X/Y X/Y X/X X/Y X/X X/X X/X X/X X/X 15/21 21 24 21 15/(23) 20 21/23 20/21 20 20/23 (19)/20 8.3/11 13 8.3 8.3 12/14 12 8.3/12 8.3/13 12/14 8.3/14 11/12 ⫺ ⫺ 11 11 ⫺ 11 ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ 11 11 11 ⫺ 11 ⫺ ⫺ ⫺ ⫺ ⫺ 26, ref.sex: X/X 58.3, ref.sex: X/Y 1076, ref.sex: X/Y 1102, ref.sex: X/Y 1500, ref.sex: X/X 1911, ref.sex: X/Y 2388, ref.sex: X/X 3742, ref.sex: X/X 3750, ref.sex: X/X 3756, ref.sex: X/X R1, ref.sex: X/X 1 ref. sex, reference molecular sex determination performed in an earlier study (Schultes 2000) with the AmpFISTR Proﬁler Plus™. Parentheses indicate alleles derived from weak signals. ⫺, no detectable ampliﬁcation result for this system. individuals who are preserved highly fragmented. Even when the amelogenin marker only showed the X-allele due to allelic dropout events, the presence of the Y-STRs still proved this individual to be male. The other way around, a female individual can be diagnosed not only by the absence of the Y-allele of amelogenin, but also by the complete absence of Y-STRs. Additionally, in most cases, at least one of the X-STRs will show a heterozygous result for females. Thus, the results of the markers in this PCR conﬁrm each other and will raise the accuracy of the sex determination of skeletal remains not amenable to morphological determination, as well as samples with poor DNA preservation and possible allelic dropouts. 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