Purification and characterization of a common soil component which inhibits the polymerase chain reaction.
DNA prepared from soil usually contains a brown-tinted inhibitor of the polymerase chain reaction (PCR) which limits the sensitivity of this technique for specific detection of microorganisms. To localize the inhibitor, soil fractions were tested for their inhibitory effect on the PCR reaction. A highly inhibitory activity, sufficient to account for the inhibition typically exhibited by soil DNA, was found to be tightly associated with the soil microorganism fraction. After cell breakage, the inhibitory material became soluble, and was not separable from DNA by standard purification procedures. A method was derived by which most of the inhibitory material could be selectively solubilized from the microorganism fraction without cell breakage, using successive washes with buffers differing in EDTA concentration. This technique was used to isolate a substance with characteristics suggesting that it is the major PCR inhibitor contaminating DNA purified from soil. It was found to be an organic, water-soluble compound of high molecular weight, and was present in a variety of soil types from different locations. It was found to be distinctly different in its solubility properties from humic and fulvic acids, and also in its FT-IR and NMR spectra. It forms a complex with protein and may inhibit the PCR reaction by an interaction with Taq DNA polymerase.
Agriculture and Agri-Food Canada, Research Branch, Ottawa, ON, Canada.
R J
RJ
Watson
B
B
Blackwell
D002170:Canada; D004247:DNA / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000187:drug effects*; D012988:Soil Microbiology; D019914:Taq Polymerase / Q000037:antagonists & inhibitors*
D004247:DNA; D019914:Taq Polymerase