Importance of DNA quality in comparative soil microbial community structure analyses

Abstract

This study assessed how different in-situ lysis soil DNA extraction methods influence the DNA yield, quality and hence the results obtained by bacterial and fungal automated ribosomal intergenic spacer analysis (ARISA). Of the methods tested in three soils, a modified hexadecyltrimethylammonium bromide-dithiotreitol (CTAB-DTT)-based method produced ⩾3 times more DNA of higher quality than the other methods (260/230nm ratios=1.64–1.82 and 260/280nm ratios=1.82–1.89 and extracts were less inhibitory of PCR). DNA extracted by this method also yielded more reproducible ARISA ribotypes (89−119 for bacteria and 48−88 for fungi; P<0.05) than DNA extracted by other methods, and consequently produced more reliable estimates of bacterial and fungal diversity in all three test soils. The significant correlations observed between the numbers of reproducible ribotypes and DNA extract 260/230nm ratios (r=0.88 and 0.72 for bacteria and fungi, respectively; P<0.001) reaffirmed the strong influence of DNA quality on the reliability of microbial diversity indices determined based on PCR-based DNA fingerprinting technique. Results of discriminant function analysis (DFA) and multivariate analysis of variances (MANOVA) performed on ARISA profiles (number and relative abundance of ribotype) revealed that the variability associated with DNA extraction methods did not exceed the biological variability among soil types; this supports the conclusion that high-quality DNA underpins DNA fingerprinting techniques.