Article

Convenient determination of DNA extraction efficiency using an external DNA recovery standard and quantitative-competitive PCR

Department of Microbiology, Miami University, Oxford, OH 45056, USA.
Journal of Microbiological Methods (Impact Factor: 2.03). 05/2004; 57(2):259-68. DOI: 10.1016/j.mimet.2004.01.013
Source: PubMed

ABSTRACT

Molecular biology techniques have advanced the field of microbial ecology through the analysis of nucleic acids. Most techniques that use DNA or RNA require their extraction from environmental matrices, which can be tedious and inefficient. While a number of extraction methods, both laboratory-based and commercially available, have been developed, none of these include a convenient method to determine extraction efficiency. We have developed an external DNA recovery standard, Lambda DNA (target DNA) contained within pBR322, allowing routine determinations of DNA recovery efficiency. Target DNA was added to sediments as whole cells, total DNA extracted using commercial DNA extraction/purification kits and the amount of target DNA recovered quantified by quantitative-competitive PCR (QC-PCR). Three commercially available kits (UltraClean Soil DNA, FastDNA SPIN and Soil Master DNA Extraction) were evaluated for recovery efficiency. Recoveries for the three kits ranged from undetectable to 43.3% with average recoveries of 14.9+/-16.0%, 28.3+/-10.5% and 2.4+/-0.1% (UltraClean, FastDNA and Soil Master, respectively). Quantification of target DNA proved robust in sediments heavily polluted with polycyclic aromatic hydrocarbons and the external recovery standard could be detected following extraction and amplification from as few as 1 x 10(3) cells added to 0.5 g sediment (wet weight). The external DNA recovery standard was also added directly to the sediment as purified plasmid DNA prior to extraction. It was recovered with similar efficiency as when added as whole cells, suggesting its usefulness in estimating DNA recovery in ribosomal DNA studies. These results show that, while the commercial kits offer expedited sample processing, the extraction efficiencies vary on a sample-by-sample basis and were <100%. Therefore, quantitative DNA studies require an estimation of DNA recovery.

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    • "This assay used species-specific primer sets targeting unique sequences found in each organism's rDNA regions without any normalization with DNA recovery efficiency (RE). In terms of sample REs, Mumy and Findlay (2004) have reported that environmental sample REs can vary among samples. In addition, Coyne et al. (2005) have pointed out that DNA recovery needs to be monitored when any DNA purification treatment is used for preparation of genomic DNA as a template for a qPCR assay. "
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