PURIFICATION OF DNA FROM ESTUARINE SEDIMENTS

DNA recovered from estuarine sediments after direct lysis of sediment microflora contains contaminants which interfere with restriction endo nuclease digestion and other enzymic manipulations. Removal of these c ontaminants has proven excessively difficult and time consuming. New p rocedures were incorporated into established direct lysis methods in o rder 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 org anic matter contents. The samples were incubated with ammonium acetate and ethidium bromide, extracted with phenol, and the aqueous DNA solu tions isopropanol precipitated. These treatments greatly reduced conta minant 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 susc eptible to restriction digestion. DNA purified using ion exchange chro matography was comparable in quality to that recovered from CsCl gradi ent ultracentrifugation, and was obtained at substantial savings in ti me and expense compared to the latter method.