, Volume 22, Issue 1, pp 71-81
Date: 03 Sep 2012

A simple extraction method suitable for PCR-based analysis of plant, fungal, and bacterial DNA

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Abstract

A simple and easy protocol for extracting high-quality DNA from microorganisms and plants is presented. The method involves inactivating proteins by using SDS/proteinase K and precipitating polysaccharides in the presence of high salt. Further purification is based on differential solubility of DNA and high-molecular-weight polysaccharides in aqueous media. The procedure does not use the toxic and potentially hazardous phenol and chloroform, and as many as 100 samples can be processed per day. Absorbency ratios (A260/A280) of 1.6–2.0 indicated a minimal presence of contaminating metabolites. The DNA was completely digested with 5 restriction enzymes:EcoR I,RsaI,TaqI,EcoR V, andHind III. PCR analysis using enterobacterial repetitive intergenic consensus (ERIC) sequence, sequence-characterized amplified region (SCAR), and random amplified microsatellite (RAMS) primers showed the DNA's compatibility with downstream applications. This procedure is applicable to a range of pathogens and plants and thus may find wide application in quarantine services and marker-assisted selection (MAS) breeding.