Optimization of random amplified polymorphic DNA techniques for use in genetic studies of Cuban triatominae Optimización de la técnica de ADN polimórfico amplificado al azar (RAPD) para su utilización en la caracterización genética de triatomíneos cubanos

Random amplified polymorphic DNA (RAPD) technique is a simple and reliable method to detect DNA polymorphism. Several factors can affect the amplification profiles, thereby causing false bands and non-reproducibility of assay. In this study, we analyzed the effect of changing the concentration of primer, magnesium chloride, template DNA and Taq DNA polymerase with the objective of determining their optimum concentration for the standardization of RAPD technique for genetic studies of Cuban Triatominae. Reproducible amplification patterns were obtained using 5 pmoL of primer, 2.5 mM of MgCl2, 25 ng of template DNA and 2 U of Taq DNA polymerase in 25 µL of the reaction. A panel of five random primers was used to evaluate the genetic variability of T. flavida. Three of these (OPA-1, OPA-2 and OPA-4) generated reproducible and distinguishable fingerprinting patterns of Triatominae. Numerical analysis of 52 RAPD amplified bands generated for all five primers was carried out with unweighted pair group method analysis (UPGMA). Jaccard's Similarity Coefficient data were used to construct a dendrogram. Two groups could be distinguished by RAPD data and these groups coincided with geographic origin, i.e. the populations captured in areas from east and west of Guanahacabibes, Pinar del Río. T. flavida present low interpopulation variability that could result in greater susceptibility to pesticides in control programs. The RAPD protocol and the selected primers are useful for molecular characterization of Cuban Triatominae.La técnica de ADN polimórfico amplificado al azar (RAPD) es un método simple para detectar el polimorfismo genético del ADN. Diferentes factores afectan los perfiles de amplificación lo que se manifiesta en la presencia de bandas falsas y en la reproducibilidad del ensayo. En nuestro trabajo analizamos los cambios de la concentración de cebador, ADN molde, cloruro de magnesio y de Taq ADN polimerasa con el objetivo de determinar su concentración optima, quedando optimizada la técnica del RAPD para su utilización en estudios genéticos de Triatomíneos cubanos. Empleando una concentración de cebador de 5 pmol, 2.5 mM de MgCl2, 25 ng de ADN molde y 2 U de Taq ADN polimerasa en 25 µL de reacción, se obtuvieron patrones de amplificación reproducibles. Un total de cinco cebadores al azar fueron usados para evaluar la variabilidad genética de T. flavida. Tres de ellos (OPA-1, OPA-2 y OPA-4) produjeron patrones distinguibles y reproducibles de triatomineos.