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. Optimization of DNA Extraction from Individual Sand Flies for PCR Amplification. Methods Protoc. 2019, 2, 36.
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. Optimization of DNA Extraction from Individual Sand Flies for PCR Amplification. Methods Protoc. 2019, 2, 36.
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 analysing parasite infection in Lutzomyia spp. by PCR [1] and, for this reason, we evaluated various modifications on a previously published protocol ([2] and Acardi personal communication). The most significant variation was the use of a different lysis buffer [3] to which added Ca2+ (buffer TESCa), because this ion protects proteinase K against autolysis, increases its thermal stability, and could have a regulatory function for its substrate-binding site [4]. Individual sand fly DNA extraction success was confirmed by amplification reactions using internal control primers that amplify a fragment of the cacophony gene [5,6]. 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.
Keywords
Sand fly; DNA extraction; Calcium; PCR, lysis buffer, Lutzomyia
Copyright:
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
