An Effective and Low-Cost Method for Genomic DNA Extraction from Cyclanthera Pedata Species (A Nutraceutical Plant) without Liquid Nitrogen for Inter Simple Sequence Repeat Analyses

Abstract

ABSTRACT: For the DNA-based study of plant species, one of the important steps is to obtain high-quality DNA. However, this is problematic when the species contains a lot of polyphenols and polysaccharides. The polysaccharides and polyphenols interfere with the activity of the Taq polymerase enzyme during the PCR reaction thereby affecting the quality of the DNA. Therefore, a method for DNA extraction from Cyclanthera pedata has been developed. The current study reveals a CTAB-based approach that is quick, dependable, and economical and is specifically designed for obtaining DNA from the Cyclanthera genus. These plant species are abundant in secondary metabolites and polysaccharides, which makes it difficult to extract DNA effectively and with a high yield. The present protocol also excludes the use of expensive liquid nitrogen, which makes it cost-friendly as well. High salt concentration (1.5 M) and 2% polyvinylpyrrolidone were used in the DNA extraction buffer to prevent the solubility of polysaccharides and polyphenols in DNA extract. In addition to these substances, protein-like various enzymes were precipitated by ammonium acetate and removed by centrifugation during the isolation process. The quality of the isolated DNA was assessed using agarose gel electrophoresis (0.8%) and quantified using an A260/A280 ratio ranging from 1.7 to 1.9, absorbance ratio >2,which indicates the extract was free of proteins, polysaccharides, and polyphenols. The extracted genomic DNA was amplified by the ISSR primer (UBC-825) and clear banding pattern were observed. This standardized protocol provides pure and high quality genomic DNA without expensive liquid nitrogen or toxic phenolic compounds. It is also suitable for routine molecular biology assays such as RAPD, SSR, restriction digestion, southern blot, and cloning techniques.