A new extraction procedure of autonomous DNA from eucaryotic cells, where DNA could be bound to proteins

The classical method for extraction of episomes from eucaryotic cells has been described by B.Hirt (1). It is based on precipitation of proteins associated to cellular DNA by sodium chloride, whereas the episomal DNA remains in the supernatant. However, this method does not allow to purify an eventual fraction of autonomous DNA which would be strongly associated with a protein as it is the case with topoisomerase II in the 'cleavable complex' formation (2). In this reaction, the DNA is cut (apparition of linear DNA in the case of close circular DNA = form IH) by the enzyme which is covalently bound to the DNA and application of the Hirt's procedure in such a case results in losing this DNA associated protein (Fig. la). Here, we describe a new and more rapid extraction method of autonomous DNA from eucaryotic cells, based on the sedimentation of nuclear DNA, which avoids this disadvantage due to the protein association. Fibroblast cell cultures (embryonic rat fibroblasts transformed by Bovine Papilloma Virus type I genome, BPV I) are incubated for one hour at 37 °C under CO2 atmosphere with different concentrations of mAMSA (as indicated in figure legend la), a cleavable complex inducing drug (3). Plates (10 cm in diameter, around 6 millions of cells) are washed twice on ice with ice-cold 0.14 m NaCl and immediately lysed with one ml of lysis buffer (50 mM Tris-HCl pH:8; 100 mM Na3-EDTA; 0.5% SDS). The lysate is collected with a tip-cut pipette in order to avoid any breakage of cellular DNA and incubated at 50-55°C at least four hours with 0.5 mg of proteinase K (Boehringer Mannheim), followed by an extraction with chloroform:isoamyl alcohol (24:1). The organic phase is counter-extracted with one ml of lysis buffer. The two aqueous phases are pooled and completed to 14 ml with the lysis buffer and centrifuged one hour at 30,000 rpm in TST 41 Kontron rotor. The cellular DNA sediments as a translucide to almost white and soft pellet. The supernatant is carefully removed and precipitated with ethanol, centrifuged in SW 28 rotor tubes and washed with 70% ethanol. The pellet is suspended in a small volume of TE buffer and loaded on a 0.8% agarose gel. DNA is transferred on a positively charged nylon membrane (Hybond-N from Amersham) in order to perform a classical Southern blotting manipulation (4).