DNA extraction

Genetic analysis of plants relies on high yield of pure DNA. Since, the leaf of Tecomella undulata accumulates large amounts of polysaccharides, polyphenols and secondary metabolites, which co-purify with genomic DNA thus leads to difficulty in extraction procedure. In the present study, experiments were carried out to extract high-quality amplifiable DNA by testing four DNA extraction methods for leaf tissues of Tecomella undulata. The key steps in the optimized protocol include increased incubation temperature, use of PVP and additives in the extraction buffer, repetition of purification step with CI and washing of DNA pellet with ethanol. The yield of DNA extracted was 209-560 µg/100mg of leaf tissue and the purity, evaluated by the ratio A260/A280, was 1.8-1.9, indicative of minimal levels of contaminating metabolites. The quality of the DNA extracted was confirmed by random amplification polymorphism DNA and by inter-simple sequence repeat amplification, proving that the DNA can be amplified via PCR.

The present study was undertaken in curry leaf plant (Murraya koenigii), one of the most important aromatic and medical plants in the world to explore the best DNA isolation protocol. Presence of abundant quantity of secondary metabolites in curry leaf plant resulted in poor quality and quantity of isolated genomic DNA. Thus, two DNA extraction protocols namely CTAB (Cetyl Trimethyl Ammonium bromide) modified method and Dellaporta modified method were evaluated for extraction of DNA. The quantity and quality of the DNA extracted was compared using UV-spectrophotometer and agarose gel electrophoresis. CTAB modified method was found to exhibit consistently positive results in terms of both quality and quantity of DNA (A260/280) as compared to Dellaporta method. The isolated DNA was highly intact and devoid of shearing. Further, this method was explored to different species of Rutaceae family, giving significant DNA yield.