Interference of humic acids and DNA extracted directly from soil in detection and transformation of recombinant DNA from bacteria and a yeast

Abstract

A two-step protocol for the extraction and purification of total DNA from soil samples was developed. Crude DNA extracts (100 μl from 5 g of soil) were contaminated with humic acids at concentrations of 0.7 to 3.3 μg/μl, depending on the type of soil extracted. The coextracted humic acid fraction of a clay silt was similar to a commercially available standard humic acid mixture, as determined by electrophoretic mobility in agarose gels, UV fluorescence, and inhibition assays with DNA-transforming enzymes. Restriction endonucleases were inhibited at humic acid concentrations of 0.5 to 17.2 μg/ml for the commercial product and 0.8 to 51.7 μg/ml for the coextracted humic acids. DNase I was less susceptible (MIC of standard humic acids, 912 μg/ml), and RNase could not be inhibited at all (MIC, >7.6 mg/ml). High inhibitory susceptibilities for humic acids were observed with Taq polymerase. For three Taq polymerases from different commercial sources, MICs were 0.08 to 0.64 μg of the standard humic acids per ml and 0.24 to 0.48 μg of the coextracted humic acids per ml. The addition of T4 gene 32 protein increased the MIC for one Taq polymerase to 5.12 μg/ml. Humic acids decreased nonradioactive detection in DNA-DNA slot blot hybridizations at amounts of 0.1 μg and inhibited transformation of competent Escherichia coli HB101 with a broad-host-range plasmid, pUN1, at concentrations of 100 μg/ml. Purification of crude DNA with ion-exchange chromatography resulted in removal of 97% of the initially coextracted humic acids. Recovery rates of soil-seeded microorganisms, carrying a mammal-derived DNA sequence as a marker gene, were 72 to 79% for Corynebacterium glutamicum ATCC 13032 pUN1, 81 to 85% for E. coli DH5-α pUN1, and 86 to 92% for Hansenula polymorpha LR9-Apr8, compared with that for DNA extracted from the respective pure cultures. Soil-extracted DNA was pure enough to detect 105 C. glutamicum pUN1 cells per g of soil by transformation of E. coli HB101, 104 cells of H. polymorpha per g of soil by slot blot DNA-DNA hybridizations, and 10 cells of H. polymorpha per g of soil by polymerase chain reaction amplification of the mammalian marker gene.