RAPD Protocol for triatominae

Abstract

ABSTRACT RAPD (Random Amplified Polymorphic DNA) technique was evaluated in silvatic and domestic I. infestans originating from Cochabamba (Bolivia). Total genomic DNA was isolated from individual legs. Three extraction steps were performed, namely lysis and deproteinisation, phenol extraction and ethanol precipation. The yield of DNA extracted was thus estimated by agarose gel electrophoresis, involving the comparison of the fluorescence intensity of ethidium stained with those of a serious of known amounts of DNA standards. Target DNA sequences were amplified by PCR in a BIOMETRA TRIO-THERMOBLOC thermal cycler. A single ten-nucleotide oligomer of random sequence (Operon Technologies Inc.) containing at least 50 % G-C served as a primer for each reaction. Amplification products were tested from purified and non-purified DNA. Different parameters such as DNA concentration, Taq DNA polymerase and MgCI2 concentration as well as the annealing temperature have been tested, and the reproducibility of the resulting optimal conditions were verified three times on the same specimens. Two brands of Taq DNA polymerase (Promega and Stratagene) were also tested. Out of 10 primers assayed, 8 gave readable patterns: primers OPA-5 and OPA-6 did not give detectable amplification products. Any change of one of the conditions retained as optimal gave variations ranging from non reproducible amplification products to the disappearence of bands or the production of a smear. Two parameters appeared to be crucial for obtaining reproducible patterns: a fixed, specific initial concentration of (i) DNA (3-5 ng) and of (ii) Taq-polymerase. Slightly lower or higher concentrations resulted in incomplete patterns or even their absence. The cost of RAPD for one individual was estimated at 1,2 US dollars (DNA extraction: 0.57, DNA quantification 0.18 and amplification 0.37), excluding the standard infrastructure needed for molecular biology. RAPD patterns allowed to readily separate domestic and silvatic ecotopes of 1. infestans, which are found nearby each another in a localized area of Cochabamba…