Optimization of DNA isolation and PCR protocol for RAPD analysis of banana / plantain (Musa spp.)

Abstract

Genetic analysis of plants relies on high yields of pure DNA samples. Here we present the optimization of DNA isolation and Polymerase chain reaction (PCR) conditions for Random Amplified Polymorphic DNA (RAPD) analysis of banana/plantain. The leaf of banana contains high level of polysaccharides, poly phenols and secondary metabolites. The extracted DNA from these cultivars when subjected to PCR is often problematic, especially when mature tissues are used for DNA extraction. In order to overcome these problems a protocol has been developed, availing on a high salt concentration and on the combination of Polyvinyl pyrrolidone (PVP) and Cetyl trimethyl ammonium bromide (CTAB) in the extraction buffer, in order to prevent the solubilization of polysaccharides and polyphenols during the DNA extraction method. It also involves successive long term chloroform: Isoamylalcohol extractions, an long term RNAse treatment with all steps carried out at Room temperature (RT). Using this method, DNA was extracted from different banana species including young leaves, old leaves, frosted old leaves and withered old leaves. The yield of DNA ranged from 1-2 μg / μl per gram of the leaf sample / tissue and the purity ratio was between 1.6-1.7 indicating minimal levels of contaminating metabolites. The technique is ideal for isolation of DNA from different plant species / cultivars and the isolated DNA were used for RAPD analysis. The optimization of RAPD protocol was based on the use of 50 ng of template DNA, higher concentration of MgCl2 (3 mM) and lower concentration of primer (0.6μM), Taq DNA polymerase (1.5 units) and an annealing temperature of 35oC, which resulted, optimal amplification. In all PCR reactions Reproducible amplifiable products were observed. Thus the results indicate that the optimized protocol for DNA isolation and PCR was applicable to plant species belonging to different genera and this process is suitable for further work on diversity analysis. Furthermore, here we used suitable DNA isolation protocol for RAPD analysis to study the genetic variation in the future in Musaceae species grown in Orissa.