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Sex Determination in Highly Fragmented Human DNA by High-Resolution Melting (HRM) Analysis

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...

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Autores principales: Álvarez-Sandoval, Brenda A., Manzanilla, Linda R., Montiel, Rafael
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4123986/
https://www.ncbi.nlm.nih.gov/pubmed/25098828
http://dx.doi.org/10.1371/journal.pone.0104629
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author Álvarez-Sandoval, Brenda A.
Manzanilla, Linda R.
Montiel, Rafael
author_facet Álvarez-Sandoval, Brenda A.
Manzanilla, Linda R.
Montiel, Rafael
author_sort Álvarez-Sandoval, Brenda A.
collection PubMed
description 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.
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spelling pubmed-41239862014-08-12 Sex Determination in Highly Fragmented Human DNA by High-Resolution Melting (HRM) Analysis Álvarez-Sandoval, Brenda A. Manzanilla, Linda R. Montiel, Rafael PLoS One Research Article 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. Public Library of Science 2014-08-06 /pmc/articles/PMC4123986/ /pubmed/25098828 http://dx.doi.org/10.1371/journal.pone.0104629 Text en © 2014 Álvarez-Sandoval et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Álvarez-Sandoval, Brenda A.
Manzanilla, Linda R.
Montiel, Rafael
Sex Determination in Highly Fragmented Human DNA by High-Resolution Melting (HRM) Analysis
title Sex Determination in Highly Fragmented Human DNA by High-Resolution Melting (HRM) Analysis
title_full Sex Determination in Highly Fragmented Human DNA by High-Resolution Melting (HRM) Analysis
title_fullStr Sex Determination in Highly Fragmented Human DNA by High-Resolution Melting (HRM) Analysis
title_full_unstemmed Sex Determination in Highly Fragmented Human DNA by High-Resolution Melting (HRM) Analysis
title_short Sex Determination in Highly Fragmented Human DNA by High-Resolution Melting (HRM) Analysis
title_sort sex determination in highly fragmented human dna by high-resolution melting (hrm) analysis
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4123986/
https://www.ncbi.nlm.nih.gov/pubmed/25098828
http://dx.doi.org/10.1371/journal.pone.0104629
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