Rapid Purification of Plasmid DNAs by Hydroxyapatite Chromatography

Abstract

A method is described for the rapid preparation of plasmid DNAs of molecular weight up to 14 x lo6.This method involves the chromatography, at room temperature, of bacterial cleared lysates on hydroxyapatite in the presence of high concentrations of phosphate and urea. All detectable protein and RNA contamination of plasmid DNA is removed by this procedure and the conformation of the plasmid DNA is unaffected. Less than 0.5 % chromosomal DNA is present in the purified preparation and even this can be removed if necessary by a simple extention of the procedure to include a heat-denaturation step. The method is extremely rapid and amenable to large-scale plasmid preparation; 5 mg ColEl DNA have been purified within 40 min. The yield of plasmid DNA is similar to that obtained with the conventional dye-centrifugation technique, however the purity is greater.The advent of recombinant DNA technology has facilitated the routine isolation of specific nucleic acid sequences of both prokaryotes and eukaryotes. This technology relies heavily on the use of vectors and for most cloning experiments bacterial plasmids are the vectors of choice. Plasmid DNA rarely constitutes as much as 50% of the total DNA present in a bacterial cell, even under growth conditions most conducive to plasmid replication [l]. Purification of plasmid DNA therefore requires the selective removal of chromosomal DNA. A very effective initial pmification is achieved by the preparation of a cleared lysate [2]. Methods for the further purification of plasmid DNA take advantage of the closed-circular nature of the DNA [3,4]. The most widely used method relies on the greater density of the closed-circular DNA relative to sheared chromosomal DNA in ethidium bromide/caesium chloride density gradients [5]. While this technique provides a high degree of purification it suffers from a number of disadvantages: it is expensive in both centrifuge time and materials, has limited capacity, and complete removal of the ethidium bromide is both difficult and tedious.In this paper we describe a purification procedure which does not depend on CsCl centrifugation and which is rapid, inexpensive and yields DNA of high purity. Our method takes advantage of the selective and reversible retention by hydroxyapatite of small double-stranded DNA molecules under conditions known to prevent the binding of all RNA and protein [6].

A rapid alkaline extraction procedure for screening recombinant plasmid DNA

“…Covalently closed circular (CCC) DNA binds a different amount of the dye than does open circular (OC) or linear DNA and is readily separated from the latter two forms of DNA (1). A second, more rapid, method for preparing plasmid DNA employs hydroxyapatite chromatography (2); this also produces highly purified DNA. However, for many purposes including analysis by gel electrophoresis, less purified DNA can be used.…”

Performance and characterization of a nanophased porous hydroxyapatite for protein chromatography

“…Besides purification of nucleic acids (Bachvarov and Ivanov, 1983;Colman et al, 1978;Freitag, 2000, 2002;Johnson and Ilan, 1983;Kuiper et al, 2002;Pakroppa et al, 1975;Shoyab and Sen, 1979;Udvardy et al, 1979), a prime application of hydroxyapatite adsorbents is the purification of antibodies at small scale as well as industrial scale (Aoyama and Chiba, 1993;Bowles et al, 1988;Giovannini and Freitag, 2001;Jungbauer et al, 1989;Luellau et al, 1998;Pavlu et al, 1986;Poiesi et al, 1989;Zola and Neoh, 1989). Another standard adsorbent for large-scale separation of antibodies in the biopharmaceutical industry is staphylococcal Protein A affinity chromatography (Boschetti and Jungbauer, 2000;Jungbauer and Wenisch, 1989).…”

Expression of the Pseudomonas Gene Coding for Carboxypeptidase G2 in Saccharomyces cerevisiae

“…Plasmids were isolated from chloramphenicol-amplified cultures of E. coli by Brij lysis (Clewell & Helinski, 1969) and caesium chloride/ethidium bromide density gradient centrifugation (Colman et al, 1978). The small-scale plasmid isolation procedure of Holmes & Quigley (1981) was used for rapid screening of transformants.…”