Interference of humic acids and DNA extracted directly from soil in detection and transformation of recombinant DNA from bacteria and a yeast.
A two-step protocol for the extraction and purification of total DNA from soil samples was developed. Crude DNA extracts (100 microliters from 5 g of soil) were contaminated with humic acids at concentrations of 0.7 to 3.3 micrograms/microliters, 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 micrograms/ml for the commercial product and 0.8 to 51.7 micrograms/ml for the coextracted humic acids. DNase I was less susceptible (MIC of standard humic acids, 912 micrograms/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 micrograms of the standard humic acids per ml and 0.24 to 0.48 micrograms of the coextracted humic acids per ml. The addition of T4 gene 32 protein increased the MIC for one Taq polymerase to 5.12 micrograms/ml. Humic acids decreased nonradioactive detection in DNA-DNA slot blot hybridizations at amounts of 0.1 micrograms and inhibited transformation of competent Escherichia coli HB101 with a broad-host-range plasmid, pUN1, at concentrations of 100 micrograms/ml. Purification of crude DNA with ion-exchange chromatography resulted in removal of 97% of the initially coextracted humic acids.(ABSTRACT TRUNCATED AT 250 WORDS)
Tebbe
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Institut für Bodenbiologie, Bundesforschungsanstalt für Landwirtschaft, Braunschweig, Germany.
Vahjen
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D007611:Aprotinin / Q000235:genetics; D001483:Base Sequence; D003352:Corynebacterium / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification*; D004274:DNA, Recombinant / Q000235:genetics; D004463:Ecology; D004926:Escherichia coli / Q000235:genetics; D006812:Humic Substances / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D010843:Pichia / Q000235:genetics; D010957:Plasmids / Q000235:genetics / Q000302:isolation & purification; D012988:Soil Microbiology; D014170:Transformation, Genetic
D004269:DNA, Bacterial; D004271:DNA, Fungal; D004274:DNA, Recombinant; D006812:Humic Substances; D007611:Aprotinin