Allelic dropout due to stochastic variation in degraded small quantity DNA appears to be one of the most serious genotyping errors. Most methods require PCR replication to address this problem. The small amounts of valuable samples are often a limitation for such replications. We report a real-time PCR-based amelogonin Y (AMELY) allele dropout estimation model in an AMEL-based gender typing. We examined 915 replicates of AMELY-positive modern male DNA with varying amounts of DNA and humic acid. A male-specific AMEL fragment (AMELy) dropped out in 143 genuine male replicates, leading to gender typing errors. By graphing a scatter plot of the crossing point versus the end cycle fluorescence of the male replicates, a standard graph model for the estimation of the AMELy allele dropout was constructed with the dropout-prone and dropout-free zones. This model was then applied to ancient DNA (aDNA) samples. Nine samples identified as female were found in the dropout-prone zone; with higher DNA concentrations, six were shifted to the dropout-free zone. Among them, two female identifications were converted to male. All the aDNA gender was confirmed by sex-determination region Y marker amplification. Our data suggest that this model could be a basic approach for securing AMELy allele dropout-safe data from the stochastic variation of degraded inhibitory DNA samples.
This is a preview of subscription content, access via your institution.
Buy single article
Instant access to the full article PDF.
Tax calculation will be finalised during checkout.
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
Tax calculation will be finalised during checkout.
Kim K, Kim KY, Jeon E, Togloom A, Cho YO, Lee MS, Lkhagvasuren G et al (2008) Technical note: improved ancient DNA purification for PCR using ion-exchange columns. Am J Phys Anthropol 136:114–121
Taberlet P, Friffin S, Goossens B, Questiau S, Manceau V, Escaravage N, Waits LP, Bouvet J (1996) Reliable genotyping of samples with very low DNA quantities using PCR. Nucleic Acids Res 24:3189–3194
Cowen S, Debenham P, Dixon A, Kutranov S, Thomson J, Way K (2011) An investigation of the robustness of the consensus method of interpreting low-template DNA profiles. Forensic Sci Int Genet 5:400–406
Gagneux P, Boesch C, Woodruff DS (1997) Microsatellite scoring errors associated with noninvasive genotyping based on nuclear DNA amplified from shed hair. Mol Ecol 6:861–868
Miller CR, Joyce P, Waits LP (2002) Assessing allelic dropout and genotype reliability using maximum likelihood. Genetics 160:357–366
Hanson EK, Ballantyne J (2005) Whole genome amplification strategy for forensic genetic analysis using single or few cell equivalents of genomic DNA. Anal Biochem 346:246–257
Strom CM, Rechitsky S (1998) Use of nested PCR to identify charred human remains and minute amounts of blood. J Forensic Sci 43:696–700
Budowle B, Eisenberg AJ, van Daal A (2009) Validity of low copy number typing and applications to forensic science. Croat Med J 50:207–217
King C, Debruyne R, Kuch M, Schwarz C, Poinar H (2009) A quantitative approach to detect and overcome PCR inhibition in ancient DNA extracts. Biotechniques 47:941–949
Kim K, Brenner CH, Mair VH, Lee KH, Kim JH et al (2010) A western Eurasian male is found in 2000-year-old elite Xiongnu cemetery in Northeast Mongolia. Am J Phys Anthropol 142:429–440
Jobling MA, Lo IC, Turner DJ et al (2007) Structural variation on the short arm of the human Y chromosome: recurrent multigene deletions encompassing amelogenin Y. Hum Mol Genet 16:307–316
Cadenas AM, Regueiro M, Gayden T, Singh N, Zhivotovsky LA, Underhill P, Herrera RJ (2007) Male amelogenin dropouts: phylogenetic context, origins and implications. Forensic Sci Int 166:155–163
Kumagai R, Sasaki Y, Tokuta T, Biwasaka H, Aoki Y (2008) DNA analysis of family members with deletion in Yp11.2 region containing amelogenin locus. Leg Med (Tokyo) 10:39–42
Yong RY, Gan LS, Chang YM, Yap EP (2007) Molecular characterization of a polymorphic 3-Mb deletion at chromosome Yp11.2 containing the AMELY locus in Singapore and Malaysia populations. Hum Genet 122:237–249
Mitchell RJ, Kreskas M, Baxter E, Buffalino L, van Oorschot RA (2006) An investigation of sequence deletions of amelogenin (AMELY), a Y-chromosome locus commonly used for gender determination. Ann Hum Biol 33:227–240
Drobnič K (2006) A new primer set in a SRY gene for sex identification. Int Congr Ser 1288:268–270
We thank Jehyeok Lee and Yeong-mi Chang for their kind assistance in administration work and purchase of laboratory materials. We also thank Jee-hyun Yoon for the maintenance of the real-time PCR system. This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-0021367).
All sampling was done according to Korean laws and ethical standards.
Conflict of interest
The authors declare that there is no conflict of interest.
About this article
Cite this article
Kim, KY., Kwon, Y., Bazarragchaa, M. et al. A real-time PCR-based amelogenin Y allele dropout assessment model in gender typing of degraded DNA samples. Int J Legal Med 127, 55–61 (2013). https://doi.org/10.1007/s00414-011-0663-5
- AMELY allele dropout
- Ancient bone
- Real-time PCR
- Melting curve analysis