SALOMON Oscar Daniel
Optimization of DNA Extraction from Individual Sand Flies for PCR Ampliﬁcation
CALIGURI LG; SANDOVAL AE; MIRANDA JC; PESSOA FA; SANTINI MS; SALOMON OD; SECUNDINO NFC; MCCARTHY CB
Methods and Protocols
Lugar: Basel; Año: 2019 vol. 2
Numerous protocols have been published for extracting DNA from phlebotomines. Nevertheless,theirsmallsizeisgenerallyanissueintermsofyield,eﬃciency,andpurity,forlarge-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.