Rapid and Efficient Isolation of High Quality DNA from Cassava (Manihot esculenta Crantz) Suitable for PCR Based Downstream Applications

摘要

The extraction of high-quality DNA from cassava leaves suitable for various molecular techniques is a challenge due to the presence of polysaccharides, proteins and polyphenols that interfere with the isolation procedures and downstream applications. This article describes a rapid and efficient procedure for isolating high yield and quality DNA from cassava leaves of six different cultivars (Kibandameno, Seveu, Mkombozi, TMS60444, TME14 and TME419). Improvement on the quantity and quality of the extracted DNA was achieved through modification of cetyl trimethylammonium bromide (CTAB) DNA extraction procedure. The modifications included addition of 20% sodium dodecyl sulfate (SDS) and 4% polyvinylpyrrolidone (PVP), use of increased concentration of ethylenediaminetetraaceticacid (EDTA) and exclusion of liquid nitrogen. The quantity and quality of extracted DNA was assessed using a spectrophotometer and agarose gel electrophoresis. The modified method in this study yielded an average amount of 2400.5 - 2919.8 ng/μl per 100 mg of leaf materials with UV absorbance ratios A260/280 of 1.81 - 1.85. Agarose gel electrophoresis (1%) illustrated intact, sharp and clear bands without degradation. The isolated DNA with this protocol served as a robust template for PCR based downstream applications of simple sequence repeats (SSR) and virus detection. The results presented in this study demonstrate the suitability of CTAB-SDS method in yielding high quality DNA from cassava leaves suitable for downstream molecular biology techniques.