PCR-SSCP comparison of 16S rDNA sequence diversity in soil DNA obtained using different isolation and purification methods

Abstract

This study compared different methods of direct DNA extraction and purification from a silt loam soil and investigated the relationship between DNA quantity and sequence diversity. Five extraction methods and four purification techniques were investigated. Quantities of DNA extracted were between 3.4 þ 0.55 and 54.3 þ 8.18 Wg g 31 (dry wt) of soil with OD 260 /OD 230 purity ratios between 0.80 and 1.15. Analysis of sequence diversity in all extracts was conducted using PCR-single strand conformation polymorphism (SSCP). Profiles generated using universal 16S rDNA primers (Com1/Com2) were found to be identical when used to amplify 16S rDNA extracted directly from soil. The genus Pseudomonas was targeted in order to reduce profile complexity, which was apparent when using universal 16S rDNA primers, and which hindered direct comparison of sequence diversity. A Pseudomonas culture library and non-cultured Pseudomonas 16S rDNA genes were used to provide a background count of Pseudomonas operational taxonomic units present in the soil. Cloning and sequencing of amplicons generated using a Pseudomonas-specific (Ps-for) and a universal 16S rDNA (Com2) primer, coupled with nested amplification (Com1/Com2 amplification from Ps-for/Ps-rev amplicons), used in conjunction with SSCP, revealed that environmental contaminants co-extracted with DNA, such as humic acid, significantly reduced primer specificity. SSCP was sensitive enough to reveal template bias in different primer sets. PCR-restriction fragment length-SSCP of Pseudomonas 16S rDNA amplified from soil-extracted DNA revealed distinct differences in sequence representation between extraction methods and showed that greater DNA yield is not synonymous with higher sequence diversity. We, therefore, suggest that DNA extractions from soil should be evaluated not only in terms of quantity and purity, but also in terms of the sequence diversity present. SSCP proved to be a valuable tool for the assessment of the methodologies commonly used in PCR-mediated microbial ecology studies. ß

Diversity of bacteria associated with Collembola – a cultivation-independent survey based on PCR-amplified 16S rRNA genes

“…These primers hybridize to phylogenetically highly conserved regions within the 16S rRNA gene and amplify products that contain the two variable regions, V4 and V5. These regions were found useful for PCR-SSCP analyses of bacterial communities in several other studies [21,38,39]. PCR with total DNA of all chosen Collembola species yielded products of the expected size (368-375 bp).…”

Phylogenetic analysis of actinobacterial populations associated with Antarctic Dry Valley mineral soils

“…This finding has been confirmed by molecular methods which have shown that the majority of microbial species, including the predominant species, are not represented in culture collections (Tindall, 2004). On the other hand, metagenomic studies may also be prone to bias which can be introduced by differences in DNA extraction (Stach et al, 2001), as well as PCR bias due to differences in primer binding (Hansen et al, 1998) or sequence heterogeneity (Suzuki et al, 1998). The main limitation of both metagenomic and traditional culturebased techniques is that one is unable to determine what role each phylotype performs within a specific ecological niche.…”

Comparative analysis of environmental DNA extraction and purification methods from different humic acid-rich soils

“…Numerous studies have been done to ascertain the most suitable DNA extraction and purification protocol from soil (e.g. Wikström et al 1996;Jackson et al 1997;Cullen and Hirsch 1998;Bürgmann et al 2001;Miller 2001;Stach et al 2001;Kauffmann et al 2004), but comparison of the results showed a number of inconsistencies. Extraction methods ranged from liquid nitrogen grinding (Volossiouk et al 1995), microwave-based rupture (Orsini and Romano-Spica 2001), enzymatic lysis (Zhou et al 1996;Stach et al 2001), to bead beating lysis (Miller et al 1999;Bürgmann et al 2001).…”