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Title: EVALUATION OF GEL FILTRATION RESINS FOR THE REMOVAL OF PCR-INHIBITORY SUBSTANCES FROM SOILS AND SEDIMENTS

Author
item Miller, Daniel

Submitted to: Journal of Microbiological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/5/2000
Publication Date: 2/1/2001
Citation: Miller, D.N. 2001. Evaluation of gel filtration resins for the removal of PCR-inhibitory substances from soils and sediments. Journal of Microbiological Methods. 44:49-58.

Interpretive Summary: Microorganisms control many processes in the environment, but very little is known about these microorganisms because of our inability to grow them. New molecular techniques have provided microbiologists tools to study microbial communities in the environment by directly studying the nucleic acids of microorganisms in environmental samples. Efficient extraction and dpurification of microbial nucleic acids from soils and sediments, however, remains a challenge. Typically, substances that inhibit molecular reactions are removed using various gel filtration resins, however, only a few resins have been rigorously evaluated. In this study, a variety of gel filtration resins (Sephadex G200 and G150; Sepharose 6B, 4B, and 2B; Bio- Gel P100, P200; and Toyopearl HW 55, HW 65, and HW 75) were evaluated for their ability to remove inhibitory substances from crude extracts of DNA from feedlot soil using gravity-flow disposable columns. Sepharose resins demonstrated the best properties for DNA purification when compared with other gel filtration resins, and Sepharose 2B was the most efficient purification resin. A method for purifying large volumes of DNA extract economically was also developed using low-cost disposable Disposaflex columns. Crude DNA extracts of microorganisms in cattle feedlot and aquifer sediments impacted by animal and human wastes were easily purified using the Disposaflex column method using two standard methods for extracting soil microbe DNA.

Technical Abstract: A variety of gel filtration resins (Sephadex G200 and G150; Sepharose 6B, 4B, and 2B; Bio-Gel P100, P200; and Toyopearl HW 55, HW 65, and HW 75) were evaluated for their efficacy in removing PCR-inhibitory substances from feedlot soil DNA crude extracts using gravity-flow disposable columns. Sepharose resins demonstrated the best properties for DNA purification when ncompared with other gel filtration resins, and Sepharose 2B was the most efficient purification resin based upon flow rate and the elution of DNA and humic acids from the columns. A method for purifying large solution volumes of DNA extract economically was also developed using low-cost disposable Disposaflex columns. Crude DNA extracts of cattle feedlot soil and aquifer sediment impacted by animal and human wastes were easily purified using the Disposaflex column method regardless of whether a gentle chemical lysis or a bead mill homogenization DNA extraction method was employed.