| Autor: |
Lorena G. Caligiuri, Adolfo E. Sandoval, Jose C. Miranda, Felipe A. Pessoa, María S. Santini, Oscar D. Salomón, Nagila F. C. Secundino, Christina B. McCarthy |
| Jazyk: |
angličtina |
| Rok vydání: |
2019 |
| Předmět: |
|
| Zdroj: |
Methods and Protocols, Vol 2, Iss 2, p 36 (2019) |
| Druh dokumentu: |
article |
| ISSN: |
2409-9279 |
| DOI: |
10.3390/mps2020036 |
| Popis: |
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. |
| Databáze: |
Directory of Open Access Journals |
| Externí odkaz: |
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