Protocol optimization for RAPD in cyanobacteria

Abstract

Random amplification of polymorphic DNA (RAPD) analysis using the polymerase chain reaction proved a useful technique in the biodiversity analysis of microorganisms but may lack reproducibility in the poorly standardized methodology. In the present investigation, the RAPD technique was optimized for characterizing cyanobacterial isolates in order to ensure its reproducibility and discriminatory power. Cyanobacterial isolates from the three genera, Anabaena, Nostoc and Calothrix were examined for the fragment patterns produced using different 10-mer RAPD primers and concentrations of different components (DNA template, primer and Taq polymerase) at two different annealing temperatures. It was observed that variations associated with all the parameters tested modified the fingerprinting pattern. For conditions not optimized, RAPD bands were faint and difficult to score. Therefore, a set of conditions for RAPD-PCR reaction has been defined, to ensure simple and fast reproducibility.