Modified Hirt procedure for rapid purification of extrachromosomal DNA from mammalian cells.

Methods for extracting extrachromosomal DNA from eukaryotic cells are important for many studies such as DNA replication and identification of certain viruses (2). The procedure described by Hirt (4), which has been widely used over the last thirty years, is laborious and time-consuming and requires phenol extraction. More rapid methods using anion-exchange chromatography (5) or silicon-based resins (3) use the alkaline lysis procedure that was originally developed for extracting plasmid DNA from bacterial cells (1). Only covalently closed circular DNA renatures correctly and stays in solution on rapid neutralization of the alkaline lysis buffer, whereas linear and open circular DNA precipitate together with the denatured proteins and chromosomal DNA (3). Thus, certain DNA replication intermediates are not recovered. This report describes a modification in which lysis at neutral pH, as in the Hirt procedure, combined with purification on a silica-gel membrane, enables the rapid recovery of linear and relaxed circular DNA along with the supercoiled form. Cells (106–107) are dispersed with trypsin, pelleted and washed in Ca2+/ Mg2+-free phosphate-buffered saline (PBS). They are resuspended in 250 μL 50 mM Tris-HCl, pH 7.5, 10 mM EDTA containing 100 μg/mL RNase A and lysed by the addition of 250 μL 1.2% sodium dodecyl sulfate (SDS). The suspension is gently mixed by inverting the tube several times and then stands for 5 min at ambient temperature. Adherent cells can be lysed directly in the culture plate after washing with PBS by overlaying them with 500 μL of a mixture of equal volumes of the above resuspension and lysis solutions. The plates are gently agitated to completely cover all cells, and the lysate is subsequently transferred to 1.5-mL microcentrifuge tubes. Cellular debris and chromosomal DNA are precipitated by the addition of 350 μL 3 M CsCl, 1 M potassium acetate and 0.67 M acetic acid. The tubes are immediately but gently mixed and placed on ice for 15 min. After centrifugation for 15 min at 14 000× g, the supernatant is loaded onto a silica gel membrane column (QIAprep Spin Column; Qiagen GmbH, Hilden, Germany). The column is washed with 750 μL 80 mM potassium acetate, 10 mM Tris-HCl, pH 7.5, 40 μM EDTA and 60% ethanol. DNA is then eluted with 50 μL water or TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). These columns are loaded, washed and eluted by centrifugation at 14 000× g or vacuum. COS cells were transfected with pSL3-EGFP, an 8.0-kb plasmid that contains the simian virus 40 (SV40) origin of replication. Twenty-four hours posttransfection, cells were suspended