Isolation of bacterial DNA from soil

Abstract

DNA has been isolated from the bacterial fraction of two soils. Numbers of bacteria, determined by fluorescence microscopy were 1.1 and 2.2·10 10 cells g −1 dry wt. The total amounts of bacterial DNA in these soils were 90 and 187 μg g −1 dry wt respectively. A modification of Marmur's method was used to isolate DNA, but difficulties in separating DNA from humic substances gave low yields and impure DNA. DNA could be partly separated from humic material in the presence of 8 m urea by ion-exchange chromatography on DEAE-Sepharose CL-6B. Final purification was obtained by chromatography on a hydroxyapatite column. When lowering the EDTA concentration in the saline-EDTA solution used for lysis, the amount of humic substances in the cell-free lysate after centrifugation was considerably decreased. The lysate could then be chromatographed directly on hydroxyapatite. Quantities up to 1.5 mg DNA high purity was isolated from 90 g wet soil (37 g dry wt). The isolated DNA was characterized by treatment with DNAse and absorption spectra. No uncommon bases were revealed by thin layer chromatography of the DNA hydrolysates. Melting curves of the isolated DNA showed a relatively broad melting profile, with half maximum hyperchromicity (T m) near 90°C. Sedimentation coefficients determined by analytical ultracentrifugation showed that the isolated DNA had a high molecular weight, ranging from 2.3 to 10.1·10 5 daltons.