Optimization of DNA Extraction from Individual Sand Flies for PCR Amplification.

Lorena G Caligiuri,Nagila F C Secundino,Adolfo E Sandoval,María S Santini,Felipe A Pessoa,Christina B Mccarthy,Jose C Miranda,Oscar D Salomón

doi:10.3390/mps2020036

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 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.

Highlights

Various protocols have been published for the extraction of DNA from phlebotomines, including methods that eliminate DNA-associated proteins by using detergents and salts [1,2,3], or with proteinase K and detergents [4], and others that add extraction steps with phenol-chloroform and precipitation with alcohol [5,6]; commercial DNA extraction kits [7,8]; and the use of Chelex-100 resin [9,10]
We found that internal control polymerase chain reaction (PCR) results were variable for individual sand flies processed with the pAC protocol
The quality and quantity of the DNA extracts were measured using an AmpliQuant AQ-07 Spectrophotometer, but we found there was no correlation between the amount of DNA quantitated and the success of the PCR reactions

Summary

Introduction

Various protocols have been published for the extraction of DNA from phlebotomines, including methods that eliminate DNA-associated proteins by using detergents and salts [1,2,3], or with proteinase K and detergents [4], and others that add extraction steps with phenol-chloroform and precipitation with alcohol [5,6]; commercial DNA extraction kits [7,8]; and the use of Chelex-100 resin [9,10]. The small size of the sand flies (around 3 mm long) can be an issue, especially in studies that require analysis on an individual basis, such as parasite infection, variability, and population genetics These large-scale individual DNA extractions using traditional methods usually yield poor results in terms of efficiency, quantity and purity, which in turn affect PCR success and DNA conservation. When we used pAC to process individual sand flies, we found that amplification was poor and inconsistent (i.e., internal control PCR results were variable) For this reason, we decided to evaluate various modifications and, as this method eliminates DNA-associated proteins with proteinase K, we focused on this first crucial step. 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

Methods

Results

Conclusion