Optimization of RAPD-PCR Conditions for the Study of Genetic Diversity in Nepalese Isolates of <i>Bacillus thuringiensis Berliner</i>

Abstract

Random amplified polymorphic DNA (RAPD) is a simple and reliable method to detect DNA polymorphism and has been used extensively for genetic diversity studies. In the present investigation the RAPD reaction and cycling conditions were optimized for generating RAPD fingerprints of ten Nepalese strains of Bacillus thuringiensis Berliner (Bt) isolated from an altitudinal range of 70 meter above sea level (masl) to 5050 masl. To determine the optimum conditions, different concentrations of MgCl₂, template DNA, Taq DNA polymerase, primer, dNTPs as well as different cycling programs were analyzed. Reproducible amplification patterns were obtained using 0.4 μM of primer, 2.5 mM of MgCl₂, 125 ng of template DNA, 0.2mM of dNTPs and 1U Taq DNA polymerase in 25 μl of the reaction volume. Cycling programs were also optimized. Out of 100 arbitrary primers screened, amplification performed with 24 primers generated the best RAPD fingerprints. The optimized RAPD-PCR conditions and the selected primers are suitable for further work on genetic diversity analysis of Nepalese isolates of Bt.

Key words: DNA fingerprint; primer; Taq DNA polymerase; template DNA

DOI: 10.3126/njst.v9i0.3171

Nepal Journal of Science and Technology 9 (2008) 91-97