A long distance RT-PCR able to amplify the Pestivirus genome

Autores
Jones, Leandro Roberto; Zandomeni, Rubén O.; Weber, Elba Laura
Año de publicación
2006
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
A method to amplify long genomic regions (up to ∼ 12.3 kb) from pestiviruses in one RT-PCR is described. The difficulty in designing conserved Pestivirus primers for the amplification of genomes from highly divergent isolates simply by means of overlapping segments is demonstrated using new bioinformatic tools. An alternative procedure consisting of optimizing the length of the genomic cDNA fragments and their subsequent amplification by polymerase chain reaction (PCR) using a limited set of specific primers is described. The amplification of long DNA fragments from a variety of sources, including genomic, mitochondrial, and viral DNAs as well as cDNA produced by reverse transcription (RT) has been achieved using this methodology, known as long distance PCR. In the case of viruses, it is necessary to obtain viral particles from infected cells prior to RT procedures. This work provides improvements in four steps of long distance RT-PCR (L-RT-PCR): (i) preparation of a viral stock, (ii) preparation of template RNA, (iii) reverse transcription and (iv) amplification of the cDNA by LD-PCR. The usefulness of L-RT-PCR is discussed in the light of current knowledge on pestivirus diversity. The genomic sequence of Singer Arg reference strain obtained using this method is presented and characterized.
Fil: Jones, Leandro Roberto. Universidad Nacional de la Patagonia Austral. Centro de Investigaciones y Transferencia Golfo San Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia Golfo San Jorge. Universidad Nacional de la Patagonia "san Juan Bosco". Centro de Investigaciones y Transferencia Golfo San Jorge; Argentina
Fil: Zandomeni, Rubén O.. Instituto Nacional de Tecnología Agropecuaria. Instituto de Microbiología y Zoología Agrícola; Argentina
Fil: Weber, Elba Laura. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina
Materia
RT-PCR
PESTIVIRUS
GENOME
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-nd/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/16792
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/16792
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling A long distance RT-PCR able to amplify the Pestivirus genomeJones, Leandro RobertoZandomeni, Rubén O.Weber, Elba LauraRT-PCRPESTIVIRUSGENOMEhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1A method to amplify long genomic regions (up to ∼ 12.3 kb) from pestiviruses in one RT-PCR is described. The difficulty in designing conserved Pestivirus primers for the amplification of genomes from highly divergent isolates simply by means of overlapping segments is demonstrated using new bioinformatic tools. An alternative procedure consisting of optimizing the length of the genomic cDNA fragments and their subsequent amplification by polymerase chain reaction (PCR) using a limited set of specific primers is described. The amplification of long DNA fragments from a variety of sources, including genomic, mitochondrial, and viral DNAs as well as cDNA produced by reverse transcription (RT) has been achieved using this methodology, known as long distance PCR. In the case of viruses, it is necessary to obtain viral particles from infected cells prior to RT procedures. This work provides improvements in four steps of long distance RT-PCR (L-RT-PCR): (i) preparation of a viral stock, (ii) preparation of template RNA, (iii) reverse transcription and (iv) amplification of the cDNA by LD-PCR. The usefulness of L-RT-PCR is discussed in the light of current knowledge on pestivirus diversity. The genomic sequence of Singer Arg reference strain obtained using this method is presented and characterized.Fil: Jones, Leandro Roberto. Universidad Nacional de la Patagonia Austral. Centro de Investigaciones y Transferencia Golfo San Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia Golfo San Jorge. Universidad Nacional de la Patagonia "san Juan Bosco". Centro de Investigaciones y Transferencia Golfo San Jorge; ArgentinaFil: Zandomeni, Rubén O.. Instituto Nacional de Tecnología Agropecuaria. Instituto de Microbiología y Zoología Agrícola; ArgentinaFil: Weber, Elba Laura. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; ArgentinaElsevier Science2006-06info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/16792Jones, Leandro Roberto; Zandomeni, Rubén O.; Weber, Elba Laura; A long distance RT-PCR able to amplify the Pestivirus genome; Elsevier Science; Journal of Virological Methods; 134; 1-2; 6-2006; 197-2040166-0934enginfo:eu-repo/semantics/altIdentifier/url/http://www.sciencedirect.com/science/article/pii/S0166093406000322info:eu-repo/semantics/altIdentifier/doi/10.1016/j.jviromet.2006.01.005info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-nd/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-03T09:45:59Zoai:ri.conicet.gov.ar:11336/16792instacron: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:34982025-09-03 09:45:59.966CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv A long distance RT-PCR able to amplify the Pestivirus genome
title A long distance RT-PCR able to amplify the Pestivirus genome
spellingShingle A long distance RT-PCR able to amplify the Pestivirus genome
Jones, Leandro Roberto
RT-PCR
PESTIVIRUS
GENOME
title_short A long distance RT-PCR able to amplify the Pestivirus genome
title_full A long distance RT-PCR able to amplify the Pestivirus genome
title_fullStr A long distance RT-PCR able to amplify the Pestivirus genome
title_full_unstemmed A long distance RT-PCR able to amplify the Pestivirus genome
title_sort A long distance RT-PCR able to amplify the Pestivirus genome
dc.creator.none.fl_str_mv Jones, Leandro Roberto
Zandomeni, Rubén O.
Weber, Elba Laura
author Jones, Leandro Roberto
author_facet Jones, Leandro Roberto
Zandomeni, Rubén O.
Weber, Elba Laura
author_role author
author2 Zandomeni, Rubén O.
Weber, Elba Laura
author2_role author
author
dc.subject.none.fl_str_mv RT-PCR
PESTIVIRUS
GENOME
topic RT-PCR
PESTIVIRUS
GENOME
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv A method to amplify long genomic regions (up to ∼ 12.3 kb) from pestiviruses in one RT-PCR is described. The difficulty in designing conserved Pestivirus primers for the amplification of genomes from highly divergent isolates simply by means of overlapping segments is demonstrated using new bioinformatic tools. An alternative procedure consisting of optimizing the length of the genomic cDNA fragments and their subsequent amplification by polymerase chain reaction (PCR) using a limited set of specific primers is described. The amplification of long DNA fragments from a variety of sources, including genomic, mitochondrial, and viral DNAs as well as cDNA produced by reverse transcription (RT) has been achieved using this methodology, known as long distance PCR. In the case of viruses, it is necessary to obtain viral particles from infected cells prior to RT procedures. This work provides improvements in four steps of long distance RT-PCR (L-RT-PCR): (i) preparation of a viral stock, (ii) preparation of template RNA, (iii) reverse transcription and (iv) amplification of the cDNA by LD-PCR. The usefulness of L-RT-PCR is discussed in the light of current knowledge on pestivirus diversity. The genomic sequence of Singer Arg reference strain obtained using this method is presented and characterized.
Fil: Jones, Leandro Roberto. Universidad Nacional de la Patagonia Austral. Centro de Investigaciones y Transferencia Golfo San Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia Golfo San Jorge. Universidad Nacional de la Patagonia "san Juan Bosco". Centro de Investigaciones y Transferencia Golfo San Jorge; Argentina
Fil: Zandomeni, Rubén O.. Instituto Nacional de Tecnología Agropecuaria. Instituto de Microbiología y Zoología Agrícola; Argentina
Fil: Weber, Elba Laura. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina
description A method to amplify long genomic regions (up to ∼ 12.3 kb) from pestiviruses in one RT-PCR is described. The difficulty in designing conserved Pestivirus primers for the amplification of genomes from highly divergent isolates simply by means of overlapping segments is demonstrated using new bioinformatic tools. An alternative procedure consisting of optimizing the length of the genomic cDNA fragments and their subsequent amplification by polymerase chain reaction (PCR) using a limited set of specific primers is described. The amplification of long DNA fragments from a variety of sources, including genomic, mitochondrial, and viral DNAs as well as cDNA produced by reverse transcription (RT) has been achieved using this methodology, known as long distance PCR. In the case of viruses, it is necessary to obtain viral particles from infected cells prior to RT procedures. This work provides improvements in four steps of long distance RT-PCR (L-RT-PCR): (i) preparation of a viral stock, (ii) preparation of template RNA, (iii) reverse transcription and (iv) amplification of the cDNA by LD-PCR. The usefulness of L-RT-PCR is discussed in the light of current knowledge on pestivirus diversity. The genomic sequence of Singer Arg reference strain obtained using this method is presented and characterized.
publishDate 2006
dc.date.none.fl_str_mv 2006-06
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/11336/16792
Jones, Leandro Roberto; Zandomeni, Rubén O.; Weber, Elba Laura; A long distance RT-PCR able to amplify the Pestivirus genome; Elsevier Science; Journal of Virological Methods; 134; 1-2; 6-2006; 197-204
0166-0934
url http://hdl.handle.net/11336/16792
identifier_str_mv Jones, Leandro Roberto; Zandomeni, Rubén O.; Weber, Elba Laura; A long distance RT-PCR able to amplify the Pestivirus genome; Elsevier Science; Journal of Virological Methods; 134; 1-2; 6-2006; 197-204
0166-0934
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/http://www.sciencedirect.com/science/article/pii/S0166093406000322
info:eu-repo/semantics/altIdentifier/doi/10.1016/j.jviromet.2006.01.005
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-nd/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-nd/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
dc.publisher.none.fl_str_mv Elsevier Science
publisher.none.fl_str_mv Elsevier Science
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 13.13397

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