Development and validation of a human DNA quantification and sex determination approach based on real time PCR followed by high resolution melting analysis

Autores
Ginart, Santiago; Alechine, Evguenia; Caputo, Mariela; Cano, H.; Corach, Daniel; Sala, Adriana Andrea
Año de publicación
2015
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
DNA quantitation is one of the most crucial factors affecting the success and quality of DNA typing by PCR. The aim of this work was to develop a DNA quantification assay to be used in routine forensic casework. It should be able to discriminate, simultaneously, the presence of male and female DNA by means of multiplex real time PCR, followed by high resolution melting analysis (HRM), including a fluorescent intercalating dye Syto 9. The approach is co-amplified fragments of a gene common to both genders, Amelogenin-Amel and a specific sequence of the human Y chromosome (HSYCS) whose melting temperature differs from Amel in 5.3–5.5 °C. Hence, it allows discriminating two peaks after HRM analysis if only male DNA is present in the sample or a single peak if only female template is present. The short length of both amplicons, 106/112 bp for Amel and 84 bp for HSYCS, facilitates quantitation and gender detection in degraded samples that characterize evidentiary material. We achieved the quantification of male, female and experimentally mixed samples with very low DNA quantities (20 pg/μl). We propose an alternative approach to commercial DNA quantitation kits. Our development showed to be fast, highly sensitive, laborsaving and cost-effective.
Fil: Ginart, Santiago. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Servicio de Huellas Digitales Genéticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina
Fil: Alechine, Evguenia. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Servicio de Huellas Digitales Genéticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina
Fil: Caputo, Mariela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Servicio de Huellas Digitales Genéticas; Argentina
Fil: Cano, H.. Laboratorio Regional de Investigación Forense; Argentina
Fil: Corach, Daniel. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Servicio de Huellas Digitales Genéticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina
Fil: Sala, Adriana Andrea. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Servicio de Huellas Digitales Genéticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina
Materia
HUMAN DNA QUANTITATION
INTERCALATING DYE
REAL TIME PCR
SEX DETERMINATION
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/117688
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/117688
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Development and validation of a human DNA quantification and sex determination approach based on real time PCR followed by high resolution melting analysisGinart, SantiagoAlechine, EvgueniaCaputo, MarielaCano, H.Corach, DanielSala, Adriana AndreaHUMAN DNA QUANTITATIONINTERCALATING DYEREAL TIME PCRSEX DETERMINATIONhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1DNA quantitation is one of the most crucial factors affecting the success and quality of DNA typing by PCR. The aim of this work was to develop a DNA quantification assay to be used in routine forensic casework. It should be able to discriminate, simultaneously, the presence of male and female DNA by means of multiplex real time PCR, followed by high resolution melting analysis (HRM), including a fluorescent intercalating dye Syto 9. The approach is co-amplified fragments of a gene common to both genders, Amelogenin-Amel and a specific sequence of the human Y chromosome (HSYCS) whose melting temperature differs from Amel in 5.3–5.5 °C. Hence, it allows discriminating two peaks after HRM analysis if only male DNA is present in the sample or a single peak if only female template is present. The short length of both amplicons, 106/112 bp for Amel and 84 bp for HSYCS, facilitates quantitation and gender detection in degraded samples that characterize evidentiary material. We achieved the quantification of male, female and experimentally mixed samples with very low DNA quantities (20 pg/μl). We propose an alternative approach to commercial DNA quantitation kits. Our development showed to be fast, highly sensitive, laborsaving and cost-effective.Fil: Ginart, Santiago. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Servicio de Huellas Digitales Genéticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: Alechine, Evguenia. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Servicio de Huellas Digitales Genéticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: Caputo, Mariela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Servicio de Huellas Digitales Genéticas; ArgentinaFil: Cano, H.. Laboratorio Regional de Investigación Forense; ArgentinaFil: Corach, Daniel. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Servicio de Huellas Digitales Genéticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: Sala, Adriana Andrea. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Servicio de Huellas Digitales Genéticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaElsevier Ireland Ltd2015-12info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/117688Ginart, Santiago; Alechine, Evguenia; Caputo, Mariela; Cano, H.; Corach, Daniel; et al.; Development and validation of a human DNA quantification and sex determination approach based on real time PCR followed by high resolution melting analysis; Elsevier Ireland Ltd; Forensic Science International: Genetics; 5; 12-2015; 269-2711875-175XCONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://www.sciencedirect.com/science/article/abs/pii/S1875176815301992info:eu-repo/semantics/altIdentifier/doi/10.1016/j.fsigss.2015.09.107info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2026-02-06T12:03:46Zoai:ri.conicet.gov.ar:11336/117688instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982026-02-06 12:03:46.962CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Development and validation of a human DNA quantification and sex determination approach based on real time PCR followed by high resolution melting analysis
title Development and validation of a human DNA quantification and sex determination approach based on real time PCR followed by high resolution melting analysis
spellingShingle Development and validation of a human DNA quantification and sex determination approach based on real time PCR followed by high resolution melting analysis
Ginart, Santiago
HUMAN DNA QUANTITATION
INTERCALATING DYE
REAL TIME PCR
SEX DETERMINATION
title_short Development and validation of a human DNA quantification and sex determination approach based on real time PCR followed by high resolution melting analysis
title_full Development and validation of a human DNA quantification and sex determination approach based on real time PCR followed by high resolution melting analysis
title_fullStr Development and validation of a human DNA quantification and sex determination approach based on real time PCR followed by high resolution melting analysis
title_full_unstemmed Development and validation of a human DNA quantification and sex determination approach based on real time PCR followed by high resolution melting analysis
title_sort Development and validation of a human DNA quantification and sex determination approach based on real time PCR followed by high resolution melting analysis
dc.creator.none.fl_str_mv Ginart, Santiago
Alechine, Evguenia
Caputo, Mariela
Cano, H.
Corach, Daniel
Sala, Adriana Andrea
author Ginart, Santiago
author_facet Ginart, Santiago
Alechine, Evguenia
Caputo, Mariela
Cano, H.
Corach, Daniel
Sala, Adriana Andrea
author_role author
author2 Alechine, Evguenia
Caputo, Mariela
Cano, H.
Corach, Daniel
Sala, Adriana Andrea
author2_role author
author
author
author
author
dc.subject.none.fl_str_mv HUMAN DNA QUANTITATION
INTERCALATING DYE
REAL TIME PCR
SEX DETERMINATION
topic HUMAN DNA QUANTITATION
INTERCALATING DYE
REAL TIME PCR
SEX DETERMINATION
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv DNA quantitation is one of the most crucial factors affecting the success and quality of DNA typing by PCR. The aim of this work was to develop a DNA quantification assay to be used in routine forensic casework. It should be able to discriminate, simultaneously, the presence of male and female DNA by means of multiplex real time PCR, followed by high resolution melting analysis (HRM), including a fluorescent intercalating dye Syto 9. The approach is co-amplified fragments of a gene common to both genders, Amelogenin-Amel and a specific sequence of the human Y chromosome (HSYCS) whose melting temperature differs from Amel in 5.3–5.5 °C. Hence, it allows discriminating two peaks after HRM analysis if only male DNA is present in the sample or a single peak if only female template is present. The short length of both amplicons, 106/112 bp for Amel and 84 bp for HSYCS, facilitates quantitation and gender detection in degraded samples that characterize evidentiary material. We achieved the quantification of male, female and experimentally mixed samples with very low DNA quantities (20 pg/μl). We propose an alternative approach to commercial DNA quantitation kits. Our development showed to be fast, highly sensitive, laborsaving and cost-effective.
Fil: Ginart, Santiago. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Servicio de Huellas Digitales Genéticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina
Fil: Alechine, Evguenia. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Servicio de Huellas Digitales Genéticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina
Fil: Caputo, Mariela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Servicio de Huellas Digitales Genéticas; Argentina
Fil: Cano, H.. Laboratorio Regional de Investigación Forense; Argentina
Fil: Corach, Daniel. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Servicio de Huellas Digitales Genéticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina
Fil: Sala, Adriana Andrea. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Servicio de Huellas Digitales Genéticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina
description DNA quantitation is one of the most crucial factors affecting the success and quality of DNA typing by PCR. The aim of this work was to develop a DNA quantification assay to be used in routine forensic casework. It should be able to discriminate, simultaneously, the presence of male and female DNA by means of multiplex real time PCR, followed by high resolution melting analysis (HRM), including a fluorescent intercalating dye Syto 9. The approach is co-amplified fragments of a gene common to both genders, Amelogenin-Amel and a specific sequence of the human Y chromosome (HSYCS) whose melting temperature differs from Amel in 5.3–5.5 °C. Hence, it allows discriminating two peaks after HRM analysis if only male DNA is present in the sample or a single peak if only female template is present. The short length of both amplicons, 106/112 bp for Amel and 84 bp for HSYCS, facilitates quantitation and gender detection in degraded samples that characterize evidentiary material. We achieved the quantification of male, female and experimentally mixed samples with very low DNA quantities (20 pg/μl). We propose an alternative approach to commercial DNA quantitation kits. Our development showed to be fast, highly sensitive, laborsaving and cost-effective.
publishDate 2015
dc.date.none.fl_str_mv 2015-12
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/117688
Ginart, Santiago; Alechine, Evguenia; Caputo, Mariela; Cano, H.; Corach, Daniel; et al.; Development and validation of a human DNA quantification and sex determination approach based on real time PCR followed by high resolution melting analysis; Elsevier Ireland Ltd; Forensic Science International: Genetics; 5; 12-2015; 269-271
1875-175X
CONICET Digital
CONICET
url http://hdl.handle.net/11336/117688
identifier_str_mv Ginart, Santiago; Alechine, Evguenia; Caputo, Mariela; Cano, H.; Corach, Daniel; et al.; Development and validation of a human DNA quantification and sex determination approach based on real time PCR followed by high resolution melting analysis; Elsevier Ireland Ltd; Forensic Science International: Genetics; 5; 12-2015; 269-271
1875-175X
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/https://www.sciencedirect.com/science/article/abs/pii/S1875176815301992
info:eu-repo/semantics/altIdentifier/doi/10.1016/j.fsigss.2015.09.107
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
application/pdf
dc.publisher.none.fl_str_mv Elsevier Ireland Ltd
publisher.none.fl_str_mv Elsevier Ireland Ltd
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
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score 13.11174

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