Modified Method of High Quality Genomic DNA Extraction from Mungbean [Vigna radiata(L.) Wilczek] Suitable for PCR Based Amplification

Abstract

Objectives: Mungbean is one among the foremost necessary pulse crops. It is necessary to extract good quality genomic DNA for molecular work in plant science. Hence, present study intended to establish a robust and cost effectively genomic DNA extraction method for mungbean. Methods/Statistical Analysis: The prevailing CTAB method for genomic DNA extraction from plant was altered by avoiding usage of liquid nitrogen or lyophilization. Genomic DNA of plant was extracted from leaves of seven mungbean genotypes by grinding in pre-heated CTAB extraction buffer. Increased concentration of CTAB, PVP, β-mercaptoethanol, use of RNase treatment and repetition of purification step with C:I after P:C:I treatment will suppress the interference of the contaminants. The effectiveness of DNA extraction protocol for PCR amplification was evaluated using SSR and RAPD markers. Findings: The quality of DNA was assured by the presence of intact bands of genomic DNA separated on 0.8% agarose gel. The yield of genomic DNA ranged from 871.6 to 1143.2 ng/μL with an average of 979.14 ng/μL. When measured spectrophotometrically, the A 260 /A 280 ratio was in the range of 1.78 to 1.86, with an average of 1.82, indicating very low levels of contamination such as polysaccharides, phenolics and protein. The results of PCR amplification using SSR and RAPD markers indicated that the DNA extracted by this modified protocol was of decent quality and appropriate for various molecular studies. This protocol is suitable for underdeveloped countries, where resources for high throughput DNA isolation are not or less available. Application/Improvement: An effective, simple and cost-effective protocol for genomic DNA extraction with good yield as well as high purity was developed for mungbean which can be applied to other pulse crops also.