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

MDPI

Lugar: Basel; Año: 2019 vol. 2

2409-9279

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.