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|Title:||Optimization of DNA Extraction from Individual Sand Flies for PCR Amplification||Authors:||Caligiuri, Lorena G
Sandoval, Adolfo E
Miranda, Jose C
Pessoa, Felipe A
Santini, María Soledad
Salomón, Oscar D
Secundino, Nagila F C
McCarthy, Christina B.
|Keywords:||Psychodidae;Extracción de ADN;Reacción en Cadena de la Polimerasa;Calcio||Issue Date:||7-May-2019||Journal:||Methods and protocols||Abstract:||
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.
|Appears in Collections:||Publicaciones INMeT|
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