Validation of laboratory methods applied to microsatellite markers

Abstract

Biodiversity information exchange and the use of genetic resources can be improved if the laboratories are able to assure their results thought suitable methodologies of validation. Aiming to define criteria which demonstrate the reliability of results in laboratories which work with DNA markers, errors associated to the DNA quantification in agarose gel, PCR repeatability and robustness using microsatellite (SSR) markers were analyzed. The error associated to DNA quantification was smaller when 100 ng of DNA were quantified, and increased for greater amounts of DNA, tending to sub estimations. Quantification limits, or the smallest quantity detected by the method used, varied markedly with small variations on the DNA amounts. Uncertainty associated to microsatellite PCR reaction was measured by its repeatability, which was affected by the quantities of DNA in the reaction. The low robustness of the microsatellite PCR reaction due to sensibility to quantities of DNA showed the priority on evaluating this parameter. The presented methodologies may be useful to laboratories working with molecular markers implementing validation procedures.