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+/-0.55 and 54.3+/-8.18 &mgr;g g(-1) (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.

Review and re-analysis of domain-specific 16S primers

“…Primer pairs were tested against DNA extracted from two Crenarchaeote and two Euryarchaeote type-strains, E. coli and two hydrothermal sediment samples from the Tokaanu and Waiotapu thermal region in New Zealand. DNA extraction was conducted using the modified Zhou method (Stach et al, 2001). …”

Polymerase chain reaction primers miss half of rRNA microbial diversity

“…Most importantly, polymerase chain reaction (PCR) primers seem to discriminate for and against certain sequences (Suzuki and Giovannoni, 1996;Polz and Cavanaugh 1998). Additionally, existing DNA extraction protocols are not 100% efficient (Stach et al, 2001). It is well known that this skews the frequency distribution of rRNA gene species among the PCR products, in clone libraries, and in eventual sequence inventories (Tiedje, 1994;Suzuki and Giovannoni, 1996;Polz and Cavanaugh 1998;Acinas et al, 2004;Caron et al, 2004;Kurata et al, 2004;Frey et al, 2006;Sipos et al, 2007).…”

Dissemination and survival of non-indigenous bacterial genomes in pristine Antarctic environments

“…Samples were kept frozen from the time of collection and stored at -80°C. Bulk DNA was extracted directly from the Antarctic Dry Valley mineral soils (Table 1) according to the modified Zhou method (Stach et al 2001). Genomic DNA concentrations ranged between 0.098 ng/g w.w. soil.…”

“…At various time intervals, bulk DNA was extracted (Stach et al 2001) from aliquots and used as template DNA in S. epidermidis-specific PCR reactions (Marti-neau et al 1996). S. epidermidis DNA could be detected in soil DNA extracts (Fig.…”