Effects of humic substances on fluorometric DNA quantification and DNA hybridization

DNA extracts from sediment and water samples are often contaminated with co extracted humic-like impurities, Estuarine humic substances and vascular pl ant extract were used to evaluate the effect of the presence of such impuri ties on DNA hybridization and quantification. The presence of humic substan ces and vascular plant extract interfered with the fluorometric measurement of DNA concentration using Hoechst dye H33258 and PicoGreen reagent. Quant ification of DNA amended with humic substances (20-80 ng/mul) using the Hoe chst dye assay was more reliable than with PicoGreen reagent. A simple proc edure was developed to improve the accuracy for determining the DNA concent ration in the presence of humic substances. In samples containing up to 80 ng/mul of humic acids, the fluorescence of the samples were measured twice: first without Hoechst dye to ascertain any fluorescence from impurities in the DNA sample, followed with Hoechst dye addition to obtain the total sam ple fluorescence. The fluorescence of the Hoechst dye-DNA complex was calcu lated by subtracting the fluorescence of the impurities from the fluorescen ce of the sample. Vascular plant extract and humic substances reduced the b inding of DNA onto the nylon membrane. Low amounts (< 2.0 mug) of humic sub stances derived from estuarine waters did not affect the binding of 100 ng of target DNA to nylon membranes. DNA samples containing 1.0 mug of humic s ubstances performed well in DNA hybridizations with DIG-labeled oliogonucle otide and chromosomal probes. Therefore, we suggest that DNA samples contai ning low concentrations of humic substances (< 20 ng/mul) could be used in quantitative membrane hybridization without further purification. (C) 2001 Elsevier Science B.V. All rights reserved.