The Purification of Plasmid DNA

The Purification of Plasmid DNA
An obvious prerequisite for cloning in plasmids is the purification of the plasmid DNA. Although a wide range of plasmid DNAs are now routinely purified the methods used are not without their problems. Undoubtedly the trickiest stage is the lysis of the host cells: both incomplete lysis and total dissolution of the cells result in greatly reduced recoveries of plasmid DNA. The ideal situation occurs when each cell Is just sufficiently broken to permit the plasmid DNA to escape without too much contaminaling chromosomal DNA. Provided the lysis is done gently most of the chromosomal DNA which is released will be of high mol. wt. and can be removed, along with cell debris, by high speed centrifugation to yield a cleared lysizre. The production of satisfactory cleared lysatas from bacteria other than E. coli and B. subtilis. particularly if large plasmids arc to be isolated, is frequently a combination of skill, luck and patience. Many methods arc available foc isolating pure plasmid DNA froen cleared lysates but only two will be described here. The first of these is die ‘classical’ method and is due to Vinograd (Radloff ci al. 1967). This method involves isopycnic centrifugation of cleared lysases in CsCI containing ethidium bromide (EtBr). EtBr binds by intercalating between the DNA base pairs and in so doing causes the DNA w unwind. A covalently closed

circular (CCC) DNA molecule such as a plasniid has no free ends and can only unwind to a limlied extent thus limiting the amount of EtBr bound. A linear DNA molecule, such as fragmented chromosomal DNA. has no such topological constraints and can therefore bmd more of the EtBr molecules. Because the density of the DNA JEt & complex decreases as more EtBr is bound, and because more EtBr can be bound to a linear molecule than a covalent cirde, the covalent circle has a higher dcnsity at saturating concentrations of EtUr. Thus covalent circles (i.e. plasmids) can be separated from linear chromosomat L)NA (Fig. 3.)).
The above method suffers from a number of disadvantages: it is expenswc in both centrifuge time and materials, has limited capacity, and complete removal of the ethidium bromide es boh difficult and tedious.
An alternative method which is both rapid and incpcnsivc is to chromatog raph the cleared lysate on hydroxylapatite (Colman ci a!. 1978). Initially conditions are chosen to ensure that all double-stranded DNA is retained by the hydroxylapatile but contaminating RNA and protein arc washed through. The DNA is then eluted from the column and cornists almost entirely (- 99%) of plasmid DNA. chromosomal DNA being physically retained presumably because of its higher mol. wi. and extended configuration.