Simple, rapid and cost-effective DNA extraction method for high quality DNA suitable for PCR based downstream application in mungbean [Vigna radiata (L.) Wilczek]

Abstract

Mungbean is a widely cultivated pulse crop. It is a source of high-quality protein for human consumption. Several new mungbean varieties have been developed employing molecular breeding tools. The quality and quantity of extracted DNA are very important for PCR amplification, particularly for the inter-simple sequence repeat-anchored resistance gene analog (ISSR-RGA) marker. In addition, reducing the time required for DNA extraction is essential in cases when large number of genotypes are analysed. Thus, the aim of this study was to compare eight combinations of two homogenization methods (manual grinding and Bullet blender® homogenizer) and four modified DNA extraction protocols. The effectiveness of DNA extraction for PCR amplification was evaluated using 3 PCR based markers, simple sequence repeat (SSR), ISSR and ISSR-RGA, on 6% polyacrylamide gel. Using homogenizer with modified protocol 2 (T6) resulted in a high DNA concentration (1032.60 ng/μl) and an A260/A280 ratio of 1.80, indicating high DNA purity. The PCR amplification of the resulting DNA with three types of molecular markers showed clear DNA bands, suggesting that DNA quality was appropriate for various molecular studies. In addition, homogenizers allowed processing of large number of samples in one go with minimal cost. These results suggest that this simple, rapid and cost-effective DNA extraction method is useful for marker-assisted breeding.