Batzer, Mark A.

Callinan, Pauline A.

Hedges, Dale J.

Shewale, Jaiprakash G.

Sinha, Sudhir K.

Walker, Jerilyn A.

Analytical Biochemistry

English

Hedges, Dale J

Walker, Jerilyn A

Callinan, Pauline A

Shewale, Jaiprakash G

Sinha, Sudhir K

Batzer, Mark A

University of Miami: Scholarship@Miami

Mobile element-based assay for human gender determination

LSU Scholarly Repository (Louisiana State Univ.)

Louisiana State University LSU Scholarly Repository Faculty Publications Department of Biological Sciences 1-1-2003 Mobile element-based assay for human gender determination Dale J. Hedges Louisiana State University Jerilyn A. Walker Louisiana State University Pauline A. Callinan Louisiana State University Jaiprakash G. Shewale ReliaGene Technologies, Inc. Sudhir K. Sinha ReliaGene Technologies, Inc. See next page for additional authors Follow this and additional works at: https://repository.lsu.edu/biosci_pubs Recommended Citation Hedges, D., Walker, J., Callinan, P., Shewale, J., Sinha, S., & Batzer, M. (2003). Mobile element-based assay for human gender determination. Analytical Biochemistry, 312 (1), 77-79. https://doi.org/10.1016/S0003-2697(02)00430-X This Article is brought to you for free and open access by the Department of Biological Sciences at LSU Scholarly Repository. It has been accepted for inclusion in Faculty Publications by an authorized administrator of LSU Scholarly Repository. For more information, please contact ir@lsu.edu. Authors Dale J. Hedges, Jerilyn A. Walker, Pauline A. Callinan, Jaiprakash G. Shewale, Sudhir K. Sinha, and Mark A. Batzer This article is available at LSU Scholarly Repository: https://repository.lsu.edu/biosci_pubs/246 Notes & TipsMobile element-based assay for human gender determinationDale J. Hedges,a Jerilyn A. Walker,a Pauline A. Callinan,a Jaiprakash G. Shewale,bSudhir K. Sinha,b and Mark A. Batzera,*a Department of Biological Sciences, Biological Computation and Visualization Center, Louisiana State University,202 Life Sciences Building, Baton Rouge, LA 70803, USAb ReliaGene Technologies, Inc., 5525 Mounes Street, Suite 101, New Orleans, LA 70123, USAReceived 20 August 2002Determination of gender from human DNA samplesis a common problem in forensic laboratories. Whileseveral PCR-based assays are currently available forhuman sex typing, each of the current approaches haslimitations. Methods based on male-specific amplifica-tion, such as the amplification of the SRY locus [1], lackan internal positive control to discriminate between fe-male DNA and male DNA which has failed to amplifyfor technical reasons. Restriction fragment lengthpolymorphism assays based on sex-specific mutations atthe ZFX/ZFY locus [2] require a second enzyme diges-tion or hybridization step following the initial PCRamplification. A recent method proposed by Cali et al.[3] based on a single adenine insertion within a tandemrepeat array at the DXYS156 locus requires access toallele detection equipment potentially unavailable toforensic labs with limited resources. The most widelyused approach is based on the Amelogenin locus, whichyields different-sized PCR amplicons for the X and Ychromosome versions of the Amelogenin gene [4].However, this method misidentifies males as females insome cases due to a deletion in the AMEL Y region [5–7]. This deletion has previously been reported to bepresent at a frequency of 0.018% in Caucasian males,1.85% among Indians, and as high as 8% in Sri Lankans[5–7]. While the frequency of the deletion is relativelylow, the crucial nature of forensic test results in cir-cumstances such as rape and prenatal gender determi-nation where there is risk for male-specific inheriteddisorders makes any source of error a legitimate causefor concern. This has led several researchers to recom-mend that Amelogenin should not be relied upon as thesole determinant of gender [5–8]. Here, we present analternative PCR method of human gender identificationbased on the presence/absence of Alu sequences.Alu elements are transposable elements which haveamplified throughout primate evolution and compriseroughly 10% of the human genome [9]. Alu insertions aregenerally considered to be homoplasy free with respect tohuman population genetics, as the probability of two Aluelements independently inserting in the same genomiclocation is extremely small [9]. The insertion of an Aluelement into a nonrecombining X–Y homologous regioncreates a way of differentiating between inserted andnoninserted chromosomes based on PCR amplicon size.While some recently integrated Alu insertions remainpolymorphic in the human population, many ultimatelyreach fixation for the presence of the Alu insertion [9].Fixed insertions on either the X or the Y chromosomeprovide a way of identifying the respective chromosome,as the inserted chromosome yields a larger fragment whenthe homologous region is amplified with PCR (Fig. 1). Byscreening X–Y homologous Alu insertions for levels ofinsertion polymorphism, we identified twomonomorphicAlu insertions thatmeet the necessary criteria for a genderdetermination assay, one fixed on the X chromosome,AluSTXa, and one fixed on theY chromosome,AluSTYa.Both of theAlu elements presumably inserted and reachedfixation in the human lineage prior to the radiation ofmodern humans from Africa. Amplification of DNAsamples from 778 diverse (African-American, European-American, and Hispanic-American) individuals of de-fined sex from paternity/identity cases for both theAluSTYa and theAluSTXa loci showed 100% accuracy ingender identification. TheDNA samples used in the studyconsisted of 389 females (278 African-American, 102European-American, and 9 Hispanic-American) and 389males (288 African-American, 90 European-American,and 11 Hispanic-American).*Corresponding author. Fax: 1-225-578-7113.E-mail address: mbatzer@lsu.edu (M.A. Batzer).Analytical Biochemistry 312 (2003) 77–79www.elsevier.com/locate/yabioANALYTICALBIOCHEMISTRY0003-2697/02/$ - see front matter  2003 Elsevier Science (USA). All rights reserved.PII: S0003 -2697 (02 )00430-XFig. 1. Schematic diagram of mobile element-based gender determination. In the diagram an Alu insertion has occurred on the Y chromosomewithin an X–Y homologous region. Once fixed in the population, the Alu insertion sequence results in a larger amplicon on the Y chromo-some, allowing for the differentiation of the sex chromosomes via PCR amplification. X chromosome-specific insertions function in the samemanner.Fig. 2. Mobile element-based gender determination. An agarose gel chromatograph from the analysis of 22 individuals using the genetic systems(A) AluSTXa and (B) AluSTYa is shown. Males are distinguished by the presence of two DNA fragments, while females have a singleamplicon. F (female) and M (male) above each sample indicate the known gender. Individual PCR amplifications were performed in 25-llreactions using 25 ng of template DNA, 0.2lM each oligonucleotide primer, 200lM deoxynucleotide-triphosphates, 1.5mM MgCl2,10mMTris–HCl (pH 8.4), and Taq DNA polymerase (1 unit). Each sample was subjected to the same amplification cycle as follows: initialdenaturation of 150 s at 94 C, 32 cycles of 1min of denaturation at 94 C, 1min at the specific annealing temperature (58 C for AluSTYaand 60 C for AluSTXa), 1min of extension at 72 C, followed by a final extension at 72 C for 10min. For analysis, 20ll of the PCR productswere fractionated on a 2% agarose gel which contained 0.25lg/ml of ethidium bromide. PCR products were visualized using ultravioletfluorescence.78 Notes & Tips / Analytical Biochemistry 312 (2003) 77–79Amplification of the loci was conducted via a PCRand fragments were resolved on a 2% agarose gel (Fig.2). The primers used for the Y insertion, AluSTYa, wereForward 50-CATGTATTTGATGGGGATAGAGG-30and Reverse 50-CCTTTTCATCCAACTACCACTGA-30,yielding an Alu-filled site (Y chromosome) fragment of528 bp and an empty site (X chromosome) fragment of199 bp. Primers for the X insertion, AluSTXa, wereForward 50-TGAAGAAATTCAGTTCATAGCTTGT-30and Reverse 50-CAGGAGATCCTGAGATTATGTGG-30, yielding an inserted (X chromosome) fragmentof 878 bp and an empty site (Y chromosome) fragmentof 556 bp. For both loci, males are distinguished ashaving two DNA amplicons present, while females haveonly a single amplicon (Fig. 2).Combining these loci together for human genderidentification will provide increased accuracy for sextyping since local deletions or other types of mutationsthat eliminate PCR would have to occur in at least twoindependent genomic locations. The speed and ease ofagarose-based genotyping due to the 300-bp differencebetween filled and empty alleles will also enhance theutility of the assay in forensic laboratories. This ap-proach should also be amenable to fluorescence-basedamplicon detection and quantitative PCR to resolvemale and female contributions to sex-mixed samples.Furthermore, similar approaches based on repetitiveelement insertions located in homologous sex chromo-some regions should be useful for gender determinationin other taxa of heterogametic sex.AcknowledgmentsThis research was supported by Award No. 2001-IJ-CX-K004 from the Office of Justice Programs, NationalInstitute of Justice, Department of Justice (M.A.B.).Points of view in this document are those of the authorsand do not necessarily represent the official position ofthe U.S. Department of Justice.References[1] A.H. Sinclair, P. Berta, M.S. Palmer, J.R. Hawkins, B.L. Griffiths,M.J. Smith, J.W. Foster, A.M. Frischauf, R. Lovell-Badge, P.N.Goodfellow, A gene from the human sex-determining regionencodes a protein with homology to a conserved DNA-bindingmotif, Nature 346 (1990) 240–244.[2] R. Reynolds, J. Varlaro, Gender determination of forensic samplesusing PCR amplification of ZFX/ZFY gene sequences, J. ForensicSci. 41 (1996) 279–286.[3] F. Cali, P. Forster, C. Kersting, M.G. Mirisola, R. DAnna, G. DeLeo, V. Romano, DXYS156: a multi-purpose short tandem repeatlocus for determination of sex, paternal and maternal geographicorigins and DNA fingerprinting, Int. J. Legal Med. 116 (2002) 133–138.[4] K.M. Sullivan, A. Mannucci, C.P. Kimpton, P. Gill, A rapid andquantitative DNA sex test: fluorescence-based PCR analysis ofX–Y homologous gene amelogenin, Biotechniques 15 (1993)636–638, 631–640.[5] F.R. Santos, A. Pandya, C. Tyler-Smith, Reliability of DNA-basedsex tests, Nat. Genet. 18 (1998) 103.[6] M. Steinlechner, B. Berger, H. Niederstatter, W. Parson, Rarefailures in the amelogenin sex test, Int. J. Legal Med. 116 (2002)117–120.[7] K. Thangaraj, A.G. Reddy, L. Singh, Is the amelogenin genereliable for gender identification in forensic casework and prenataldiagnosis?, Int. J. Legal Med. 116 (2002) 121–123.[8] B. Brinkmann, Is the amelogenin sex test valid?, Int. J. Legal Med.116 (2002) 63.[9] M.A. Batzer, P.L. Deininger, Alu repeats and human genomicdiversity, Nat. Rev. Genet. 3 (2002) 370–379.Notes & Tips / Analytical Biochemistry 312 (2003) 77–79 79

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Mobile element-based assay for human gender determination

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