Optimization of DNA extraction from individual sand flies for PCR amplification

Autores
Caligiuri, L. G.; Sandoval, A. E.; Miranda, J. C.; Pessoa, F. A.; Santini, M. S.; Salomón, O. D.; Secundino, N. F. C.; McCarthy, C. B.
Año de publicación
2019
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Numerous protocols have been published for extracting DNA from phlebotomines. Nevertheless, their small size is generally an issue in terms of yield, efficiency, and purity, for large-scale individual sand fly DNA extractions when using traditional methods. Even though this can be circumvented with commercial kits, these are generally cost-prohibitive for developing countries. We encountered these limitations when analyzing field-collected Lutzomyia spp. by polymerase chain reaction (PCR) and, for this reason, we evaluated various modifications on a previously published protocol, the most significant of which was a different lysis buffer that contained Ca2+ (buffer TESCa). This ion protects proteinase K against autolysis, increases its thermal stability, and could have a regulatory function for its substrate-binding site. Individual sand fly DNA extraction success was confirmed by amplification reactions using internal control primers that amplify a fragment of the cacophony gene. To the best of our knowledge, this is the first time a lysis buffer containing Ca2+ has been reported for the extraction of DNA from sand flies.
Fil: Caligiuri, L. G. Universidad Nacional de La Plata. Facultad de Ciencias Exactas (UNLP); Argentina
Fil: Sandoval, A. E. Instituto Nacional de Tecnología Industrial (INTI); Argentina
Fil: Miranda, J. C. Fundação Oswaldo Cruz; Brasil
Fil: Pessoa, F. A. Fundação Oswaldo Cruz; Brasil
Fil: Santini, M. S. Administración Nacional de Laboratorios e Institutos de Salud Dr. Carlos G. Malbrán (ANLIS); Argentina
Fil: Salomón, O. D. Instituto Nacional de Medicina Tropical; Argentina
Fil: Secundino, N. F. C. Fundação Oswaldo Cruz; Brasil
Fil: McCarthy, C. B. Universidad Nacional de La Plata. Facultad de Ciencias Exactas (UNLP); Argentina
Fuente
Methods and Protocols, 20(2)
Materia
ADN
PCR
Biotecnología
Calcio
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by/4.0/
Repositorio
Repositorio Institucional del Instituto Nacional de Tecnología Industrial (INTI)
Institución
Instituto Nacional de Tecnología Industrial
OAI Identificador
nuevadc:Sandoval2019DNA_pdf
Acceder
id RIINTI_4aa671fb6f2f33acaf9fe4b6867b4008
oai_identifier_str nuevadc:Sandoval2019DNA_pdf
network_acronym_str RIINTI
repository_id_str
network_name_str Repositorio Institucional del Instituto Nacional de Tecnología Industrial (INTI)
spelling Optimization of DNA extraction from individual sand flies for PCR amplificationCaligiuri, L. G.Sandoval, A. E.Miranda, J. C.Pessoa, F. A.Santini, M. S.Salomón, O. D.Secundino, N. F. C.McCarthy, C. B.ADNPCRBiotecnologíaCalcioNumerous protocols have been published for extracting DNA from phlebotomines. Nevertheless, their small size is generally an issue in terms of yield, efficiency, and purity, for large-scale individual sand fly DNA extractions when using traditional methods. Even though this can be circumvented with commercial kits, these are generally cost-prohibitive for developing countries. We encountered these limitations when analyzing field-collected Lutzomyia spp. by polymerase chain reaction (PCR) and, for this reason, we evaluated various modifications on a previously published protocol, the most significant of which was a different lysis buffer that contained Ca2+ (buffer TESCa). This ion protects proteinase K against autolysis, increases its thermal stability, and could have a regulatory function for its substrate-binding site. Individual sand fly DNA extraction success was confirmed by amplification reactions using internal control primers that amplify a fragment of the cacophony gene. To the best of our knowledge, this is the first time a lysis buffer containing Ca2+ has been reported for the extraction of DNA from sand flies.Fil: Caligiuri, L. G. Universidad Nacional de La Plata. Facultad de Ciencias Exactas (UNLP); ArgentinaFil: Sandoval, A. E. Instituto Nacional de Tecnología Industrial (INTI); ArgentinaFil: Miranda, J. C. Fundação Oswaldo Cruz; BrasilFil: Pessoa, F. A. Fundação Oswaldo Cruz; BrasilFil: Santini, M. S. Administración Nacional de Laboratorios e Institutos de Salud Dr. Carlos G. Malbrán (ANLIS); ArgentinaFil: Salomón, O. D. Instituto Nacional de Medicina Tropical; ArgentinaFil: Secundino, N. F. C. Fundação Oswaldo Cruz; BrasilFil: McCarthy, C. B. Universidad Nacional de La Plata. Facultad de Ciencias Exactas (UNLP); ArgentinaMDPI2019info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfSandoval2019DNA.pdfhttps://app.inti.gob.ar/greenstone3/sites/localsite/collect/nuevadc/index/assoc/Sandoval.dir/doc.pdfMethods and Protocols, 20(2)reponame:Repositorio Institucional del Instituto Nacional de Tecnología Industrial (INTI)instname:Instituto Nacional de Tecnología Industrialenginfo:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by/4.0/openAccess2025-09-29T15:02:04Znuevadc:Sandoval2019DNA_pdfinstacron:INTIInstitucionalhttps://app.inti.gob.ar/greenstone3/biblioOrganismo científico-tecnológicohttps://argentina.gob.ar/intihttps://app.inti.gob.ar/greenstone3/oaiserver?verb=Identifypfalcato@inti.gob.arArgentinaopendoar:2025-09-29 15:02:05.287Repositorio Institucional del Instituto Nacional de Tecnología Industrial (INTI) - Instituto Nacional de Tecnología Industrialfalse
dc.title.none.fl_str_mv Optimization of DNA extraction from individual sand flies for PCR amplification
title Optimization of DNA extraction from individual sand flies for PCR amplification
spellingShingle Optimization of DNA extraction from individual sand flies for PCR amplification
Caligiuri, L. G.
ADN
PCR
Biotecnología
Calcio
title_short Optimization of DNA extraction from individual sand flies for PCR amplification
title_full Optimization of DNA extraction from individual sand flies for PCR amplification
title_fullStr Optimization of DNA extraction from individual sand flies for PCR amplification
title_full_unstemmed Optimization of DNA extraction from individual sand flies for PCR amplification
title_sort Optimization of DNA extraction from individual sand flies for PCR amplification
dc.creator.none.fl_str_mv Caligiuri, L. G.
Sandoval, A. E.
Miranda, J. C.
Pessoa, F. A.
Santini, M. S.
Salomón, O. D.
Secundino, N. F. C.
McCarthy, C. B.
author Caligiuri, L. G.
author_facet Caligiuri, L. G.
Sandoval, A. E.
Miranda, J. C.
Pessoa, F. A.
Santini, M. S.
Salomón, O. D.
Secundino, N. F. C.
McCarthy, C. B.
author_role author
author2 Sandoval, A. E.
Miranda, J. C.
Pessoa, F. A.
Santini, M. S.
Salomón, O. D.
Secundino, N. F. C.
McCarthy, C. B.
author2_role author
author
author
author
author
author
author
dc.subject.none.fl_str_mv ADN
PCR
Biotecnología
Calcio
topic ADN
PCR
Biotecnología
Calcio
dc.description.none.fl_txt_mv Numerous protocols have been published for extracting DNA from phlebotomines. Nevertheless, their small size is generally an issue in terms of yield, efficiency, and purity, for large-scale individual sand fly DNA extractions when using traditional methods. Even though this can be circumvented with commercial kits, these are generally cost-prohibitive for developing countries. We encountered these limitations when analyzing field-collected Lutzomyia spp. by polymerase chain reaction (PCR) and, for this reason, we evaluated various modifications on a previously published protocol, the most significant of which was a different lysis buffer that contained Ca2+ (buffer TESCa). This ion protects proteinase K against autolysis, increases its thermal stability, and could have a regulatory function for its substrate-binding site. Individual sand fly DNA extraction success was confirmed by amplification reactions using internal control primers that amplify a fragment of the cacophony gene. To the best of our knowledge, this is the first time a lysis buffer containing Ca2+ has been reported for the extraction of DNA from sand flies.
Fil: Caligiuri, L. G. Universidad Nacional de La Plata. Facultad de Ciencias Exactas (UNLP); Argentina
Fil: Sandoval, A. E. Instituto Nacional de Tecnología Industrial (INTI); Argentina
Fil: Miranda, J. C. Fundação Oswaldo Cruz; Brasil
Fil: Pessoa, F. A. Fundação Oswaldo Cruz; Brasil
Fil: Santini, M. S. Administración Nacional de Laboratorios e Institutos de Salud Dr. Carlos G. Malbrán (ANLIS); Argentina
Fil: Salomón, O. D. Instituto Nacional de Medicina Tropical; Argentina
Fil: Secundino, N. F. C. Fundação Oswaldo Cruz; Brasil
Fil: McCarthy, C. B. Universidad Nacional de La Plata. Facultad de Ciencias Exactas (UNLP); Argentina
description Numerous protocols have been published for extracting DNA from phlebotomines. Nevertheless, their small size is generally an issue in terms of yield, efficiency, and purity, for large-scale individual sand fly DNA extractions when using traditional methods. Even though this can be circumvented with commercial kits, these are generally cost-prohibitive for developing countries. We encountered these limitations when analyzing field-collected Lutzomyia spp. by polymerase chain reaction (PCR) and, for this reason, we evaluated various modifications on a previously published protocol, the most significant of which was a different lysis buffer that contained Ca2+ (buffer TESCa). This ion protects proteinase K against autolysis, increases its thermal stability, and could have a regulatory function for its substrate-binding site. Individual sand fly DNA extraction success was confirmed by amplification reactions using internal control primers that amplify a fragment of the cacophony gene. To the best of our knowledge, this is the first time a lysis buffer containing Ca2+ has been reported for the extraction of DNA from sand flies.
publishDate 2019
dc.date.none.fl_str_mv 2019
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 Sandoval2019DNA.pdf
https://app.inti.gob.ar/greenstone3/sites/localsite/collect/nuevadc/index/assoc/Sandoval.dir/doc.pdf
identifier_str_mv Sandoval2019DNA.pdf
url https://app.inti.gob.ar/greenstone3/sites/localsite/collect/nuevadc/index/assoc/Sandoval.dir/doc.pdf
dc.language.none.fl_str_mv eng
language eng
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by/4.0/
openAccess
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by/4.0/
openAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv MDPI
publisher.none.fl_str_mv MDPI
dc.source.none.fl_str_mv Methods and Protocols, 20(2)
reponame:Repositorio Institucional del Instituto Nacional de Tecnología Industrial (INTI)
instname:Instituto Nacional de Tecnología Industrial
reponame_str Repositorio Institucional del Instituto Nacional de Tecnología Industrial (INTI)
collection Repositorio Institucional del Instituto Nacional de Tecnología Industrial (INTI)
instname_str Instituto Nacional de Tecnología Industrial
repository.name.fl_str_mv Repositorio Institucional del Instituto Nacional de Tecnología Industrial (INTI) - Instituto Nacional de Tecnología Industrial
repository.mail.fl_str_mv pfalcato@inti.gob.ar
_version_ 1844623655798046720
score 12.559606

Publicaciones similares