PURIFICATION OF DNA FROM ESTUARINE SEDIMENTS

DNA recovered from estuarine sediments after direct lysis of sediment microflora contains contaminants which interfere with restriction endonuclease digestion and other enzymic manipulations. Removal of these contaminants has proven excessively difficult and time consuming. New procedures were incorporated into established direct lysis methods in order to improve the quality of DNA recovered from sediments. Crude DNA preparations were recovered from estuarine sediment samples spanning a broad range of median grain sizes, % silt and clay contents, and organic matter contents. The samples were incubated with ammonium acetate and ethidium bromide, extracted with phenol, and the aqueous DNA solutions isopropanol precipitated. These treatments greatly reduced contaminant quantity, as determined by spectrophotometric analysis, but did not yield restriction digestible DNA. This partially purified DNA was further purified using ion exchange chromatography, yielding DNA susceptible to restriction digestion. DNA purified using ion exchange chromatography was comparable in quality to that recovered from CsCl gradient ultracentrifugation, and was obtained at substantial savings in time and expense compared to the latter method.