SIMPLE AND RAPID METHOD FOR THE ISOLATION OF GENOMIC DNA FROM SOIL SAMPLES OF EXTREME HABITATSKODAPARTHI ASHWITHA, PAVAN KUMAR PINDI AND A. SWAROOPA RANIAbstract Extraction mixture, Genomic DNA, Humic acid, Metagenomic, Molecular methods, PCR Abstract Because the composition of different extreme habitats varies with respect to their matrix, organic and inorganic compounds and biotic factors, standardization of total DNA extraction technique is desirable. Improved DNA extraction techniques could help to ensure a metagenomic library that adequately represents the entire communitys genome without inhibitory substances. Since most analyses of soil biodiversity and functions assume that the DNA extracted represents the microbial community in the soil, but subsequent interpretations are limited by the amount of DNA recovered from the soil. Unfortunately, extraction methods do not provide uniform and correct metagenomic DNA, and as a consequence, accurate species distributions cannot be determined. Already existed protocols are extremely time consuming and complex processes. The present study dealt with the rapid and simple isolation of heterologous genomic DNA by manually prepared single extraction lysis mixture which included extraction buffer, lysozyme and SDS followed by incubation at optimal temperature of 55 oC and then the DNA obtained from the lyses soil sample was purified by strong acid (3M HCl) and low base (0.1M NaOH) treatment for humic acid removal followed by purification using phenol-chloroform-isoamyl alcohol-ethanol purification for protein and other contaminants, it gives high DNA yields. Purified crude DNA was also evaluated for percent recovery, fragment size, restriction Digestion and PCR amplification. An addition advantage of this method is that only 500mg of soil and 2hrs of time is very much enough to isolate a large quantity of heterologous genomic DNA from soil sample.
Enter your contact information below to receive full paper.
|