Standardization of DNA extraction protocol and screening of begomovirus in chilli and tomato plants

Abstract

Extraction of high-quality genomic DNA is an important requisite for a number of molecular- and genome-based studies. Isolation of good quantity and quality of genomic DNA from plant materials is a major concern for molecular biologists as many of them contain large amount of polysaccharides, phenolics and secondary metabolites. The presence of polyphenols and polysaccharides in plants results in a brown–dark brown-coloured, contaminated DNA which is sticky and viscous in nature. In our present study, we performed DNA isolation from two plants (tomato and chilli) using Dellaporta, modified CTAB, phenanthroline-based, CaCl2-based DNA extraction protocols (with and without high-salt Tris-EDTA buffer treatment). For chilli plants, Dellaporta method with high-salt TE buffer treatment was found to be better than other methods, and for tomato plants modified CTAB and phenanthroline-based methods without high-salt TE treatment were better. To further confirm the applicability, total DNA was isolated from 99 chilli samples using Dellaporta method with high-salt TE treatment. The plants were then screened for geminivirus and 49 were found to be positive. Furthermore, one of the PCR products of Deng primer was cloned and analysis of the clones showed the presence of insert, thus confirming the applicability of the DNA extraction method for detection of virus, PCR and cloning.