Protocol optimization for genomic DNA extraction and RAPD-PCR in mosquito larvae (Diptera: Culicidae)

PCR based molecular markers are powerful tools for the analysis of genetic diversity for which isolation of good quality genomic DNA is essential. This paper presents a comparison of three DNA extraction methods for 1, 5 and 10 larvae of Aedes aegypti. First method employing DNAzol® involves simple and one step protocol for DNA isolation where maximum purity (A260/A280 1.9) was obtained with single larva which decreased by increasing the number of mosquito larvae. DNA isolation with DNeasy® kit according to the procedure recommended by Qiagen provided the most consistent and reproducible results with high A260/A280 ratio (>1.9) with single and five larvae but resulted in slight smearing with ten larvae. However, SDS based DNA extraction protocol is cost effective and provides purity level comparable to DNeasy® kit as observed by the genomic DNA profile of 1% agarose electrophoresis. The effect of temperature and incubation time was also investigated on the DNA yield. Moreover, the DNA yield extracted by SDS method was 1.4 times higher than other methods and was found suitable for RAPD analysis which could be used for the identification and genetic diversity evaluation of Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus larvae.