Effect of Proteinase-K on Genomic DNA Extraction from Gram-positive Strains

Abstract

Direct extraction of DNA from natural environment and clinical samples has become a useful alternative for the phylogenetic identification and in situ detection of individual microbial cells without cultivation. In this study, three different Gram positive microorganisms ( B. cereus, B. subtilis, and S. aureus ) were chosen for genomic DNA extraction. High salt SDS (Sodium Dodesyl Sulfate) based extraction method was followed to extract genomic DNA with addition of three different lysis protocols to observe the effect of proteinase-K on total genomic DNA yield, lysis steps were carried with SDS, SDS with 3 μl proteinase-K and SDS with 6μl proteinase-K. High molecular weight intact DNA bands were observed only for Bacillus subtilis when the extraction procedure was carried out in presence of SDS, SDS with proteinase-K (3μl) and SDS with increased amount of proteinase-K (6μl). In presence of SDS and increased amount of proteinase-K (6μl) the mean value of DNA concentration for Bacillus cereus , Bacillus subtilis , and Staphylococcus aureus were found to be 1.53±0.15, 1.36±0.10 and 1.65±0.10 μg/μl respectively. However, in absence of proteinase-K, the mean values of DNA concentration were found to be decreased (1.28±0.10, 1.34±0.15, 1.23±0.10 μg/μl for B. cereus, B. subtilis , and S. aureus respectively) for all these stains. Although in case of B. subtilis the overall effect of proteinase-K was not found to be significant in terms of DNA concentration and DNA band intensity, however, for B. cereus , and S. aureus sharp decrease in total extracted DNA concentration was observed suggesting the increased lysis effect of proteinase-K on the thick peptidoglycan layer of Gram-positive cell wall such as B. cereus , and S. aureus . Key words : Extraction; Genomic DNA; Lysis buffer; Gram positive organism. DOI: http://dx.doi.org/10.3329/sjps.v4i1.8867 SJPS 2011; 4(1): 53-57