Optimization of DNA Extraction from Individual Sand Flies for PCR Amplification

Mostrar todas las versiones(3)

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Caligiuri, Lorena Gisel, Sandoval, Adolfo E, Miranda, José C., Pessoa, Felipe A., Santini, María Soledad, Salomón, Oscar D., Secundino, Nagila F. C., McCarthy, Christina Beryl
Formato: Articulo
Lenguaje:Inglés
Publicado: 2019
Materias:
Ciencias Médicas
Bioquímica
Sand fly
DNA extraction
Calcium
PCR
Lysis buffer
Lutzomyia
Acceso en línea:http://sedici.unlp.edu.ar/handle/10915/107515
http://europepmc.org/backend/ptpmcrender.fcgi?accid=PMC6632178&blobtype=pdf
Aporte de:
SEDICI (UNLP) de Universidad Nacional de La Plata
id I19-R120-10915-107515
record_format dspace
institution Universidad Nacional de La Plata
institution_str I-19
repository_str R-120
collection SEDICI (UNLP)
language Inglés
topic Ciencias Médicas
Bioquímica
Sand fly
DNA extraction
Calcium
PCR
Lysis buffer
Lutzomyia
spellingShingle Ciencias Médicas
Bioquímica
Sand fly
DNA extraction
Calcium
PCR
Lysis buffer
Lutzomyia
Caligiuri, Lorena Gisel
Sandoval, Adolfo E
Miranda, José C.
Pessoa, Felipe A.
Santini, María Soledad
Salomón, Oscar D.
Secundino, Nagila F. C.
McCarthy, Christina Beryl
Optimization of DNA Extraction from Individual Sand Flies for PCR Amplification
topic_facet Ciencias Médicas
Bioquímica
Sand fly
DNA extraction
Calcium
PCR
Lysis buffer
Lutzomyia
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.
format Articulo
Articulo
author Caligiuri, Lorena Gisel
Sandoval, Adolfo E
Miranda, José C.
Pessoa, Felipe A.
Santini, María Soledad
Salomón, Oscar D.
Secundino, Nagila F. C.
McCarthy, Christina Beryl
author_facet Caligiuri, Lorena Gisel
Sandoval, Adolfo E
Miranda, José C.
Pessoa, Felipe A.
Santini, María Soledad
Salomón, Oscar D.
Secundino, Nagila F. C.
McCarthy, Christina Beryl
author_sort Caligiuri, Lorena Gisel
title Optimization of DNA Extraction from Individual Sand Flies for PCR Amplification
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
publishDate 2019
url http://sedici.unlp.edu.ar/handle/10915/107515
http://europepmc.org/backend/ptpmcrender.fcgi?accid=PMC6632178&blobtype=pdf
work_keys_str_mv AT caligiurilorenagisel optimizationofdnaextractionfromindividualsandfliesforpcramplification
AT sandovaladolfoe optimizationofdnaextractionfromindividualsandfliesforpcramplification
AT mirandajosec optimizationofdnaextractionfromindividualsandfliesforpcramplification
AT pessoafelipea optimizationofdnaextractionfromindividualsandfliesforpcramplification
AT santinimariasoledad optimizationofdnaextractionfromindividualsandfliesforpcramplification
AT salomonoscard optimizationofdnaextractionfromindividualsandfliesforpcramplification
AT secundinonagilafc optimizationofdnaextractionfromindividualsandfliesforpcramplification
AT mccarthychristinaberyl optimizationofdnaextractionfromindividualsandfliesforpcramplification
bdutipo_str Repositorios
_version_ 1764820443223556098

Ejemplares similares