Comparison of Phenol-Chloroform and CTAB Assay for DNA Extraction from Polysaccharides-Rich Simarouba Glauca DC Applying Modified CTAB Method

Abstract

Different methods available for extraction of plant genomic DNA suffers from one or more drawbacks including compromised quality, quantity and many more. The extraction of high-quality DNA Simarouba glauca DC is notoriously troublesome due to the high contents of polysaccharides and different secondary metabolites. Herein, we aimed to develop a modified CTAB extraction method to isolate DNA from tissues containing high levels of polysaccharides. The principle modifications currently employed for DNA extraction involved the use of higher CTAB concentration with higher levels of β- mercaptoethanol. Additionally, higher concentrations of sodium chloride and potassium acetate were added simultaneously with absolute ice chilled isopropanol for the precipitation of DNA free from polysaccharides. Absorbance at 260 and 280 nm, respectively, were estimated to check the quality and quantity of the extracted DNA sample. It was found that the presently describe method had good quality, presented mean concentration value 233.93 ± 61.52 ng/μl (260/280 = 1.47 ± 0.2). In contrast to modified CTAB method, others method showed mean concentration of 701.52 ± 80,46, 673.01 ± 120.21, 431.96 ± 81.88 ng/μl with absorbance ratios of 1.02 ± 0.08, 0.89 ± 0.06 and 1.00 ± 0.07, respectively. Qualitative assessment of the extracted DNA was checked by Polymerase Chain reaction (PCR) and double digestion of the DNA sample. This method solved the problems of viscous DNA contaminated with polysaccharides, suitable for downstream applications such as restriction, cloning, genetic mapping or marker-assisted breeding.