Novel perspectives to purify genomic DNA from high humic acid content and contaminated soils

Abstract

Numerous soil nucleic acid isolation protocols published during the last decades reflect difficulties to remove all traces of DNA-coprecipitated contaminants such as humic acids and pollutants. The aim of this work was to optimize a rapid and innovative DNA isolation and purification procedure from high humic acid content and contaminated soil. Two types of vitamins, pyridoxal hydrochloride and thiamine hydrochloride, were used as PCR inhibitor removals. The combination of vitamins with a “salting-out” with potassium chloride (KCl) during the optimization experiments enabled the recovery of PCR-ready DNA extracts from polluted soils with high organic matter contents. To investigate the extent of this purification method, genomic nucleic acids were also extracted from soil samples artificially supplemented with 10% (w/w) of pure humic acids. Successful PCR amplifications confirmed the high purity of the DNA recovered from all samples. The use of cetyl trimethylammonium bromide (CTAB) with vitamins instead of KCl prevented DNA losses and also allowed the recovery of high quality genomic DNA from the different soils, depending on lysis buffer pH adjustment. The application of this protocol followed by bacterial diversity analysis by PCR–TGGE demonstrated that this method could be applied to a bacterial molecular diversity analysis from a wide variety of soils. The major advantage of the described procedure is the inherent short processing time with no preliminary soil washing requirement or additional purification step.