|Bolton, Harvey, Jr.|
Submitted to: Journal of Microbiological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/1/1996
Publication Date: N/A
Citation: Interpretive Summary: To monitor specific organisms in soil it is often necessary to use techniques that identify DNA. For example if we wanted to track the growth or presence of organisms that converted fertilizer nitrogen to nitrate we may try to detect specific DNA related to these organisms. These techniques are useful for analyzing contaminated soils as well. The problem is that to detect specific DNA whole soil has to be extracted which usually results in a contaminated mix of complex substances. Our objective was to develop a method of 'cleaning up' these soil extracts to more easily detect the DNA of interest. We were successful in developing an electroelution technique to remove complex compounds that interfere with the detection of specific DNA from the organisms of interest. This technique will make it possible to detect with more confidence beneficial organisms for contaminated soil cleanup and for studying organisms that may play a role in efficient agricultural systems.
Technical Abstract: The application of nucleic acid techniques to detect, identify, and monitor specific genes or organisms in soils or sediments is often complicated by the inhibitory effects of humic compounds that copurify with nucleic acids. A rapid electroelution technique was developed to separate inhibitory compounds from extracts of soil DNA and RNA. This technique was used in conjunction with PCR to detect nifH, terrestrial ammonia-oxidizer (TAO) 16S rRNA genes and TAO 16S rRNA from a variety of surface soils and contaminated sediments. After electroelution of crude nucleic acid extracts, PCR sensitivity was increased up to a factor of 10E4 relative to DNA templates that had not been electroeluted. Without electroelution of crude DNA extracts, target genes often remained undetected. Likewise, electroelution of crude RNA extracts increased RT PCR sensitivity (for TAO 16S rRNA) by a factor of 10E3 relative to RNA extracts that had not been further purified. The electroelution technique will therefore be useful for rendering environmental nucleic acids extracted from soil and sediment more amenable to PCR methods and nucleic acid analysis.