title: Sex determination in highly fragmented human DNA by high-resolution melting (HRM) analysis.
abstract: Sex identification in ancient human remains is a common problem especially if the skeletons are sub-adult, incomplete or damaged. In this paper we propose a new method to identify sex, based on real-time PCR amplification of small fragments (61 and 64 bp) of the third exon within the amelogenin gene covering a 3-bp deletion on the AMELX-allele, followed by a High Resolution Melting analysis (HRM). HRM is based on the melting curves of amplified fragments. The amelogenin gene is located on both chromosomes X and Y, showing dimorphism in length. This molecular tool is rapid, sensitive and reduces the risk of contamination from exogenous genetic material when used for ancient DNA studies. The accuracy of the new method described here has been corroborated by using control samples of known sex and by contrasting our results with those obtained with other methods. Our method has proven to be useful even in heavily degraded samples, where other previously published methods failed. Stochastic problems such as the random allele drop-out phenomenon are expected to occur in a less severe form, due to the smaller fragment size to be amplified. Thus, their negative effect could be easier to overcome by a proper experimental design.
authors.affiliation: Laboratorio Nacional de Genómica para la Biodiversidad, Unidad de Genómica Avanzada, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Irapuato, Guanajuato, Mexico.
authors.forename: Brenda A
authors.initials: BA
authors.lastname: Álvarez-Sandoval
authors.affiliation: Instituto de Investigaciones Antropológicas, Universidad Nacional Autónoma de México, Mexico City, Mexico.
authors.forename: Linda R
authors.initials: LR
authors.lastname: Manzanilla
authors.affiliation: Laboratorio Nacional de Genómica para la Biodiversidad, Unidad de Genómica Avanzada, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Irapuato, Guanajuato, Mexico.
authors.forename: Rafael
authors.initials: R
authors.lastname: Montiel
mesh_terms: D000328:Adult; D053523:Amelogenin / Q000235:genetics*; D041321:Chromosomes, Human, X / Q000235:genetics*; D041322:Chromosomes, Human, Y / Q000235:genetics*; D004247:DNA / Q000235:genetics*; D005260:Female; D006801:Humans; D008297:Male; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012732:Sex Determination Analysis / Q000379:methods*
chemical_list: C507199:AMELX protein, human; D053523:Amelogenin; D004247:DNA
references.citation: Forensic Sci Int Genet. 2012 Jan;6(1):97-101
references.pmid: 21458395
references.citation: J Forensic Sci. 2012 Jan;57(1):24-34
references.pmid: 21939443
references.citation: Int J Legal Med. 2013 Jan;127(1):55-61
references.pmid: 22237796
references.citation: Forensic Sci Med Pathol. 2010 Dec;6(4):255-60
references.pmid: 20369311
references.citation: Forensic Sci Int. 2008 Aug 25;180(1):1-9
references.pmid: 18692331
references.citation: Int J Legal Med. 2009 Jan;123(1):25-33
references.pmid: 18553095
references.citation: J Forensic Sci. 2003 Nov;48(6):1309-13
references.pmid: 14640276
references.citation: J Hum Genet. 2001;46(5):263-6
references.pmid: 11355016
references.citation: Nucleic Acids Res. 2003 Nov 15;31(22):e136
references.pmid: 14602929
references.citation: J Forensic Sci. 2003 Mar;48(2):263-73
references.pmid: 12664981
references.citation: Biotechniques. 1997 Jan;22(1):176-81
references.pmid: 8994665
references.citation: J Forensic Leg Med. 2013 Jul;20(5):387-91
references.pmid: 23756502
references.citation: Naturwissenschaften. 1991 Jun;78(6):266-7
references.pmid: 1922388
references.citation: Am J Phys Anthropol. 2009 Sep;140(1):143-8
references.pmid: 19358295
references.citation: Int J Legal Med. 1994;106(4):190-3
references.pmid: 8038111
references.citation: J Forensic Sci. 2007 Mar;52(2):264-70
references.pmid: 17316220
references.citation: Cleft Palate J. 1990 Apr;27(2):121-30
references.pmid: 2187631
references.citation: PLoS One. 2013;8(1):e52524
references.pmid: 23372650
references.citation: Forensic Sci Int. 2000 Sep 11;113(1-3):87-90
references.pmid: 10978606
references.citation: Forensic Sci Int. 2005 Jan 29;147(2-3):181-4
references.pmid: 15567624
references.citation: Mol Biol Evol. 2007 Apr;24(4):998-1004
references.pmid: 17255122
references.citation: J Forensic Sci. 1989 Sep;34(5):1206-13
references.pmid: 2809544
references.citation: Forensic Sci Int. 2008 Oct 25;181(1-3):53.e1-4
references.pmid: 18814978
references.citation: Forensic Sci Int. 2012 Jun 10;219(1-3):290.e1-7
references.pmid: 22209293
references.citation: Am J Phys Anthropol. 2010 Oct;143(2):279-84
references.pmid: 20853481
references.citation: Genomics. 1991 Feb;9(2):264-9
references.pmid: 2004775
references.citation: J Forensic Sci. 2012 Mar;57(2):295-305
references.pmid: 22211822
references.citation: J Forensic Leg Med. 2011 May;18(4):173-6
references.pmid: 21550567
references.citation: EMBO J. 2009 Sep 2;28(17):2494-502
references.pmid: 19661919
references.citation: J Forensic Sci. 2011 Mar;56(2):289-96
references.pmid: 21210801
references.citation: Am J Hum Genet. 1992 Feb;50(2):303-16
references.pmid: 1734713
references.citation: J Forensic Leg Med. 2008 May;15(4):223-6
references.pmid: 18423354
references.citation: J Forensic Leg Med. 2011 Jan;18(1):14-7
references.pmid: 21216373
references.citation: Nat Genet. 1998 Feb;18(2):103
references.pmid: 9462733
references.citation: Forensic Sci Int. 2005 Jan 29;147(2-3):113-7
references.pmid: 15567614
references.citation: Dentomaxillofac Radiol. 2012 Mar;41(3):197-202
references.pmid: 22116135
references.citation: Surg Radiol Anat. 2007 Feb;29(1):9-13
references.pmid: 17171233
references.citation: Biotechniques. 1997 Jan;22(1):130-1, 134-8
references.pmid: 8994660
references.citation: J Am Dent Assoc. 1997 Jul;128(7):1021-5
references.pmid: 9231609
references.citation: Genomics. 1989 Feb;4(2):162-8
references.pmid: 2737677
references.citation: Int J Legal Med. 2002 Apr;116(2):117-20
references.pmid: 12056519
references.citation: Int J Dev Biol. 1995 Feb;39(1):127-33
references.pmid: 7626398
references.citation: Am J Phys Anthropol. 2003 Aug;121(4):337-41
references.pmid: 12884315
references.citation: Anthropol Anz. 2000 Mar;58(1):1-8
references.pmid: 10816779
references.citation: Nature. 2003 Jun 19;423(6942):825-37
references.pmid: 12815422
references.citation: Biotechniques. 2002 Apr;32(4):790-2, 794-6
references.pmid: 11962601
references.citation: J Virol Methods. 2006 Mar;132(1-2):146-53
references.pmid: 16293321
references.citation: Forensic Sci Int. 1998 Jul 6;95(1):39-45
references.pmid: 9718670
references.citation: J Forensic Odontostomatol. 2007 Jun;25(1):7-11
references.pmid: 17577972
references.citation: Clin Chem. 2006 Mar;52(3):494-503
references.pmid: 16423901
references.citation: J Forensic Leg Med. 2013 Oct;20(7):906-11
references.pmid: 24112343
references.citation: J Forensic Leg Med. 2013 Oct;20(7):855-60
references.pmid: 24112336
references.citation: Hum Genet. 2005 Apr;116(5):395-401
references.pmid: 15726419
references.citation: Am J Med Genet. 1991 Jun 15;39(4):472-3
references.pmid: 1877627
references.citation: Int J Legal Med. 2002 Apr;116(2):121-3
references.pmid: 12056520
references.citation: J Forensic Sci. 1998 Sep;43(5):959-63
references.pmid: 9729811
references.citation: Forensic Sci Int. 2010 Jun 15;199(1-3):111.e1-3
references.pmid: 20395082
references.citation: Croat Med J. 2003 Jun;44(3):273-80
references.pmid: 12808718
references.citation: Int J Legal Med. 2008 Jul;122(4):333-5
references.pmid: 18351373
references.citation: J Forensic Leg Med. 2010 Jul;17(5):243-6
references.pmid: 20569949
references.citation: Forensic Sci Int. 2008 Oct 25;181(1-3):54.e1-5
references.pmid: 18829191
references.citation: J Forensic Sci. 2003 Nov;48(6):1280-7
references.pmid: 14640271
references.citation: Nucleic Acids Res. 1996 Aug 15;24(16):3189-94
references.pmid: 8774899
references.citation: Anal Biochem. 1997 Feb 15;245(2):154-60
references.pmid: 9056205
references.citation: J Forensic Sci. 2012 Jan;57(1):172-5
references.pmid: 22074354
references.citation: Anthropol Anz. 2010;68(1):21-9
references.pmid: 20954453
references.citation: J Forensic Sci. 2000 Jan;45(1):61-7
references.pmid: 10641920
references.citation: Am J Phys Anthropol. 1996 Feb;99(2):231-8
references.pmid: 8967324
references.citation: Int J Legal Med. 2011 Sep;125(5):669-73
references.pmid: 20652712
references.citation: J Forensic Sci. 2010 Jan;55(1):149-52
references.pmid: 20002272
references.citation: Gene. 1995 Dec 29;167(1-2):327-32
references.pmid: 8566801
references.citation: Forensic Sci Int. 1991 Jan-Feb;49(1):81-8
references.pmid: 2032670
references.citation: Biotechniques. 1993 Oct;15(4):636-8, 640-1
references.pmid: 8251166
references.citation: Forensic Sci Int. 1992 Jan;52(2):143-8
references.pmid: 1601346
references.citation: Hum Mol Genet. 2007 Feb 1;16(3):307-16
references.pmid: 17189292
references.citation: Mol Ecol. 1997 Sep;6(9):861-8
references.pmid: 9301074
references.citation: J Forensic Sci. 1996 Sep;41(5):865-7
references.pmid: 8789849
references.citation: Int J Legal Med. 2013 Sep;127(5):1039-44
references.pmid: 23344564
references.citation: Forensic Sci Int. 2011 Feb 25;205(1-3):36-9
references.pmid: 20605076
references.citation: Forensic Sci Int. 2010 Apr 15;197(1-3):120.e1-5
references.pmid: 20083365
references.citation: Forensic Sci Med Pathol. 2011 Sep;7(3):233-47
references.pmid: 21080110
references.citation: Int J Legal Med. 1997;110(6):312-5
references.pmid: 9387013
references.citation: Genomics. 1992 Sep;14(1):203-5
references.pmid: 1427830
references.citation: J Forensic Sci. 2001 Sep;46(5):1159-63
references.pmid: 11569559
references.citation: Hum Biol. 2001 Oct;73(5):689-713
references.pmid: 11758690
references.citation: Forensic Sci Int. 2012 Jan 10;214(1-3):210.e1-4
references.pmid: 21871745
references.citation: Forensic Sci Int Genet. 2009 Mar;3(2):74-9
references.pmid: 19215875
references.citation: J Forensic Sci. 1994 Jul;39(4):1047-56
references.pmid: 8064263
references.citation: Homo. 2010 Aug;61(4):253-70
references.pmid: 20638062
references.citation: Forensic Sci Int. 2001 Mar 1;117(1-2):23-30
references.pmid: 11230943
references.citation: J Forensic Leg Med. 2013 Jul;20(5):471-9
references.pmid: 23756517
references.citation: Forensic Sci Int. 2005 Jan 29;147(2-3):135-9
references.pmid: 15567617
references.citation: Sud Med Ekspert. 2003 Jul-Aug;46(4):19-22
references.pmid: 12939838