COMBINED LIPID DNA EXTRACTION METHOD FOR ENVIRONMENTAL-SAMPLES/

Previously, separate methods have been developed for the extraction an d purification of lipids and DNA from soils and sediments. This paper describes a new method for the isolation of both lipids and DNA from t he same environmental sample. This combined method is based on the Bli gh and Dyer lipid extraction technique. Upon phase separation, lipids partition into the organic phase and DNA partitions into the aqueous p hase. DNA extraction and recovery from the solid phase is necessary un der certain conditions. Preliminary experiments performed with P-32-la beled DNA in the absence of soil showed that greater than 98% of the t otal DNA was present in the aqueous phase after the modified Bligh and Dyer extraction. Analysis of the DNA by polyacrylamide gel electropho resis and autoradiography demonstrated that no degradation of DNA occu rred during the lipid extraction procedure. Lipid extraction of lyophi lized cells showed that DNA was released from Pseudomonas putida and B acillus subtilis cells corresponding to 26+/-5 and 14+/-4% of the theo retical DNA yield, respectively. The combined lipid/DNA extraction met hod was applied to both lyophilized cells and wet cells added to soil. Analysis by DNA:DNA hybridization showed that approx. 40-50% of the D NA from cells added to soil was recovered after lipid extraction relat ive to samples treated only with conventional DNA extraction. Estimati on of cell number per gram soil based on either lipid or DNA analysis showed good agreement with actual numbers added based on plate counts of the inocula. DNA extracts from samples which had been lipid-extract ed also had lower amounts of humic material. Although some DNA was not recovered after lipid extraction, that which was recovered was of suf ficiently high quality for hybridization analysis, This method shows u tility for the co-recovery of both lipids and DNA from a single sample ; this is particularly useful when a small sample size is all that is available or procurable.