A procedure for extracting plasmid DNA from bacterial cells is described. The method is simple enough to permit the analysis by gel electrophoresis of 100 or more clones per day yet yields plasmid DNA which is pure enough to be digestible by restriction enzymes. The principle of the method is selective alkaline denaturation of high molecular weight chromosomal DNA while covalently closed circular DNA remains double-stranded. Adequate pH control is accomplished without using a pH meter. Upon neutralization, chromosomal DNA renatures to form an insoluble clot, leaving plasmid DNA in the supernatant. Large and small plasmid DNAs have been extracted by this method.

A rapid alkaline extraction procedure for screening recombinant plasmid DNA

Aprocedure forextracting plasmid DNAfrombacterial cellsis described. Themethodissimpleenough topermit theanalysis bygel electrophoresis of100ormoreclonesperdayyetyieldsplasmid DNAwhichis pureenough tobedigestible byrestriction enzymes.Theprinciple ofthe methodisselective alkaline denaturation ofhighmolecular weightchromosomal DNAwhilecovalently closedcircular DNAremains double-stranded. Adequate pHcontrol isaccomplished without usinga pHmeter.Uponneutralization, chromosomal DNArenatures toformaninsoluble clot,leaving plasmid DNAin thesupernatant. Largeandsmallplasmid DNAshavebeenextracted bythis method.

Arapid alkaline extraction procedure forscreening recombinant plasmid DNA

The Huckel theory, with an extended basis set consisting of 2s and 2p carbon and 1s hydrogen orbitals, with inclusion of overlap and all interactions, yields a good qualitative solution of most hydrocarbon conformational problems. Calculations have been performed within the same parametrization for nearly all simple saturated and unsaturated compounds, testing a variety of geometries for each. Barriers to internal rotation, ring conformations, and geometrical isomerism are among the topics treated. Consistent σ and π charge distributions and overlap populations are obtained for aromatics and their relative roles discussed. For alkanes and alkenes charge distributions are also presented. Failures include overemphasis on steric factors, which leads to some incorrect isomerization energies; also the failure to predict strain energies. It is stressed that the geometry of a molecule appears to be its most predictable quality.

An Extended Hückel Theory. I. Hydrocarbons

Procedures are described for the detection and isolation of plasmids of various sizes (2.6 to 350 megadaltons) that are harbored in species of Agrobacterium, Rhizobium, Escherichia, Salmonella, Erwinia, Pseudomonas, and Xanthomonas. The method utilized the molecular characteristics of covalently closed circular deoxyribonucleic acid (DNA) that is released from cells under conditions that denature chromosomal DNA by using alkaline sodium dodecyl sulfate (pH 12.6) at elevated temperatures. Proteins and cell debris were removed by extraction with phenol-chloroform. Under these conditions chromosomal DNA concentrations were reduced or eliminated. The clarified extract was used directly for electrophoretic analysis. These procedures also permitted the selective isolation of plasmid DNA that can be used directly in nick translation, restriction endonuclease analysis, transformation, and DNA cloning experiments.

Rapid procedure for detection and isolation of large and small plasmids.

https://www.cell.com/cell/pdf/0092-8674(84)90301-5.pdf

Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis

The interaction of platinum and palladium complexes with closed and nicked circular and linear DNAs was investigated by a variety of methods. Cationic metal complexes containing flat, aromatic ligands, such as 2,2',2''-terpyridine, o-phenanthroline, and 2,2'-bipyridine, interfere with the usual fluorescence enhancement of ethidium bromide by competing for intercalation sites on calf-thymus DNA. Metal complexes having kinetically exchangable ligands, including the antitumor drugs cis-[(NH3)2PtCl2] and [(en)PtC12], inhibit noncompetitively the DNA-associated ethidium fluorescence enhancement by binding covalently to the bases and blocking potential intercalation sites. Only the metallointercalators were capable of altering the DNA duplex winding, as judged by the effects of these reagents upon the electrophoretic mobility and sedimentation behavior of PM-2 DNAs. Long-term (t greater than 120 h) interactions of metal complexes with PM-2 DNAs I, I0, and II, corresponding to superhelical, closed relaxed, and nicked circles, respectively, showed that covalent binding occurs the most readily to DNA I, possibly because of the presence of underwound duplex regions in this tightly wound superhelical DNA. The active antitumor drugs cis-[(NH3)2PtC12] and [(en)PtC12] bind covalently to DNA I under conditions where the inactive trans- [(NH3)2PtC12] does not. Most of the complexes studied were capable of producing chain scissions in PM-2DNA I. Exceptions are the kinetically inert complexes [(bipy)Pt(en)]2+ and (terpy)Pt(SCH2CH2OH)]+, suggesting that covalent binding might be a prerequisite for nicking.

Binding of platinum and palladium metallointercalation reagents and antitumor drugs to closed and open DNAs.

Closed duplex (cd) DNA was first detected in 1963 in extracts of polyoma virus (49, 149), and the unique structural properties of this nucleic acid were recognized in 1965 by Vinograd and co-workers (130). The special properties of cdDNA may be placed into three logical categories: (a) the covalent continuity of the individual strands introduces a conservation condition, expressed as the interstrand winding or linking number; (b) the structure of the DNA is severely constrained as a consequence of this requirement, and consequently these molecules possess aspects of tertiary and even higher-order structure; and (c) the chemical reactivity of cdDNA is altered (either enhanced or diminished) toward reagents that alter the duplex winding. In some cases, cdDNA is also especially reactive toward reagents that are normally specific for single-stranded (ss) DNA. The chemical and biological properties of cdDNA have been the subject of several recent reviews (11, 12, 38a, 65, 81).

Structure and reactions of closed duplex dna

The antitumor drug cis-dichlorodiammineplatinum(II) (cis-DDP) and the inactive trans isomer bind and produce cooperative changes in closed and nicked circular duplex DNA's. Covalent binding of both platinum complexes to the closed circular DNA alters the degree of supercoiling, presumably by disrupting and unwinding the double helix. Electron micrographs show the platinated DNA's to be shortened by up to 50 percent of their original length. At similar ratios of bound platinum per nucleotide, the electrophoretic mobilities of the DNA's in gels containing the dye ethidium bromide are the same for both isomers. The only detectable difference in the binding of the two platinum isomers is an increase in the electrophoretic mobility in nondye gels of closed circular DNA having small amounts of bound cis-DDP that is not apparent for the trans complex.

https://science.sciencemag.org/content/sci/203/4384/1014.full.pdf

William R. Bauer

Papers

Binding of platinum and palladium metallointercalation reagents and antitumor drugs to closed and open DNAs.

Structure and reactions of closed duplex dna

Binding of cis- and trans-dichlorodiammineplatinum(II) to DNA: evidence for unwinding and shortening of the double helix

Supercoiling in closed circular DNA: dependence upon ion type and concentration.

Nucleosome structural changes due to acetylation.