High-resolution melting analysis for bird sexing: a successful
Autor(a) principal: | |
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Data de Publicação: | 2013 |
Outros Autores: | , , , , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Texto Completo: | http://hdl.handle.net/10348/4297 |
Resumo: | High-resolution melting (HRM) analysis is a very attractive and flexible advanced post-PCR method with high sensitivity/ specificity for simple, fast and cost-effective genotyping based on the detection of specific melting profiles of PCR products. Next generation real-time PCR systems, along with improved saturating DNA-binding dyes, enable the direct acquisition of HRM data after quantitative PCR. Melting behaviour is particularly influenced by the length, nucleotide sequence and GC content of the amplicons. This method is expanding rapidly in several research areas such as human genetics, reproductive biology, microbiology and ecology/conservation of wild populations. Here we have developed a successful HRM protocol for avian sex identification based on the amplification of sexspecific CHD1 fragments. The melting curve patterns allowed efficient sexual differentiation of 111 samples analysed (plucked feathers, muscle tissues, blood and oral cavity epithelial cells) of 14 bird species. In addition, we sequenced the amplified regions of the CHD1 gene and demonstrated the usefulness of this strategy for the genotype discrimination of various amplicons (CHD1Z and CHD1W), which have small size differences, ranging from 2 bp to 44 bp. The established methodology clearly revealed the advantages (e.g. closed-tube system, high sensitivity and rapidity) of a simple HRM assay for accurate sex differentiation of the species under study. The requirements, strengths and limitations of the method are addressed to provide a simple guide for its application in the field of molecular sexing of birds. The high sensitivity and resolution relative to previous real-time PCR methods makes HRM analysis an excellent approach for improving advanced molecular methods for bird sexing. |
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High-resolution melting analysis for bird sexing: a successfulBirdsCHD1 geneHigh-resolution melting analysisMolecular sex identificationReal-time PCRHigh-resolution melting (HRM) analysis is a very attractive and flexible advanced post-PCR method with high sensitivity/ specificity for simple, fast and cost-effective genotyping based on the detection of specific melting profiles of PCR products. Next generation real-time PCR systems, along with improved saturating DNA-binding dyes, enable the direct acquisition of HRM data after quantitative PCR. Melting behaviour is particularly influenced by the length, nucleotide sequence and GC content of the amplicons. This method is expanding rapidly in several research areas such as human genetics, reproductive biology, microbiology and ecology/conservation of wild populations. Here we have developed a successful HRM protocol for avian sex identification based on the amplification of sexspecific CHD1 fragments. The melting curve patterns allowed efficient sexual differentiation of 111 samples analysed (plucked feathers, muscle tissues, blood and oral cavity epithelial cells) of 14 bird species. In addition, we sequenced the amplified regions of the CHD1 gene and demonstrated the usefulness of this strategy for the genotype discrimination of various amplicons (CHD1Z and CHD1W), which have small size differences, ranging from 2 bp to 44 bp. The established methodology clearly revealed the advantages (e.g. closed-tube system, high sensitivity and rapidity) of a simple HRM assay for accurate sex differentiation of the species under study. The requirements, strengths and limitations of the method are addressed to provide a simple guide for its application in the field of molecular sexing of birds. The high sensitivity and resolution relative to previous real-time PCR methods makes HRM analysis an excellent approach for improving advanced molecular methods for bird sexing.Blackwell Publishing Ltd2015-03-17T14:02:39Z2013-01-01T00:00:00Z2013info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/10348/4297engdoi: 10.1111/1755-0998.12081metadata only accessinfo:eu-repo/semantics/openAccessMorinha, FranciscoTravassos, PauloSeixas, FernandaSantos, NunoSargo, RobertoSousa, LuísMagalhães, PaulaCabral, João AlexandreBastos, Estelareponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2024-02-02T12:37:15Zoai:repositorio.utad.pt:10348/4297Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-20T02:01:42.666592Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse |
dc.title.none.fl_str_mv |
High-resolution melting analysis for bird sexing: a successful |
title |
High-resolution melting analysis for bird sexing: a successful |
spellingShingle |
High-resolution melting analysis for bird sexing: a successful Morinha, Francisco Birds CHD1 gene High-resolution melting analysis Molecular sex identification Real-time PCR |
title_short |
High-resolution melting analysis for bird sexing: a successful |
title_full |
High-resolution melting analysis for bird sexing: a successful |
title_fullStr |
High-resolution melting analysis for bird sexing: a successful |
title_full_unstemmed |
High-resolution melting analysis for bird sexing: a successful |
title_sort |
High-resolution melting analysis for bird sexing: a successful |
author |
Morinha, Francisco |
author_facet |
Morinha, Francisco Travassos, Paulo Seixas, Fernanda Santos, Nuno Sargo, Roberto Sousa, Luís Magalhães, Paula Cabral, João Alexandre Bastos, Estela |
author_role |
author |
author2 |
Travassos, Paulo Seixas, Fernanda Santos, Nuno Sargo, Roberto Sousa, Luís Magalhães, Paula Cabral, João Alexandre Bastos, Estela |
author2_role |
author author author author author author author author |
dc.contributor.author.fl_str_mv |
Morinha, Francisco Travassos, Paulo Seixas, Fernanda Santos, Nuno Sargo, Roberto Sousa, Luís Magalhães, Paula Cabral, João Alexandre Bastos, Estela |
dc.subject.por.fl_str_mv |
Birds CHD1 gene High-resolution melting analysis Molecular sex identification Real-time PCR |
topic |
Birds CHD1 gene High-resolution melting analysis Molecular sex identification Real-time PCR |
description |
High-resolution melting (HRM) analysis is a very attractive and flexible advanced post-PCR method with high sensitivity/ specificity for simple, fast and cost-effective genotyping based on the detection of specific melting profiles of PCR products. Next generation real-time PCR systems, along with improved saturating DNA-binding dyes, enable the direct acquisition of HRM data after quantitative PCR. Melting behaviour is particularly influenced by the length, nucleotide sequence and GC content of the amplicons. This method is expanding rapidly in several research areas such as human genetics, reproductive biology, microbiology and ecology/conservation of wild populations. Here we have developed a successful HRM protocol for avian sex identification based on the amplification of sexspecific CHD1 fragments. The melting curve patterns allowed efficient sexual differentiation of 111 samples analysed (plucked feathers, muscle tissues, blood and oral cavity epithelial cells) of 14 bird species. In addition, we sequenced the amplified regions of the CHD1 gene and demonstrated the usefulness of this strategy for the genotype discrimination of various amplicons (CHD1Z and CHD1W), which have small size differences, ranging from 2 bp to 44 bp. The established methodology clearly revealed the advantages (e.g. closed-tube system, high sensitivity and rapidity) of a simple HRM assay for accurate sex differentiation of the species under study. The requirements, strengths and limitations of the method are addressed to provide a simple guide for its application in the field of molecular sexing of birds. The high sensitivity and resolution relative to previous real-time PCR methods makes HRM analysis an excellent approach for improving advanced molecular methods for bird sexing. |
publishDate |
2013 |
dc.date.none.fl_str_mv |
2013-01-01T00:00:00Z 2013 2015-03-17T14:02:39Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://hdl.handle.net/10348/4297 |
url |
http://hdl.handle.net/10348/4297 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
doi: 10.1111/1755-0998.12081 |
dc.rights.driver.fl_str_mv |
metadata only access info:eu-repo/semantics/openAccess |
rights_invalid_str_mv |
metadata only access |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.publisher.none.fl_str_mv |
Blackwell Publishing Ltd |
publisher.none.fl_str_mv |
Blackwell Publishing Ltd |
dc.source.none.fl_str_mv |
reponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação instacron:RCAAP |
instname_str |
Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
instacron_str |
RCAAP |
institution |
RCAAP |
reponame_str |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
collection |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
repository.name.fl_str_mv |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
repository.mail.fl_str_mv |
|
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1799137101759905792 |