%0 Journal Article
%T Optimization of DNA Extraction from Individual Sand Flies for PCR Amplification
%J Methods and Protocols | An Open Access Journal from MDPI
%D 2019
%R https://doi.org/10.3390/mps2020036
%X 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 Ca 2+ (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 Ca 2+ has been reported for the extraction of DNA from sand flies. View Full-Tex
%U https://www.mdpi.com/2409-9279/2/2/36