Biomaterial Database

Ethidium bromide-mediated renaturation of denatured closed circular DNAs: mechanistic aspects and fractionation of DNA on a molecular weight basis. 1989

P P Lau, and X G Zhou, and H B Gray

Department of Biochemical and Biophysical Sciences, University of Houston, TX 77204.

When closed circular duplex DNAs are exposed to alkali in the presence of ethidium bromide, from 0 to 100% of the DNA can be recovered as the fully base-paired duplex (native) form upon neutralization of the solutions. The fraction of native DNA depends on the concentration of ethidium bromide, time of incubation, ionic strength and temperature of the solutions before neutralization as well as the molecular weight and superhelix density of the DNA. Limiting ethidium concentrations exist below and above which 0 and 100% of the DNA, respectively, is recovered as native material under a given set of incubation conditions regardless of the length of time of incubation before neutralization. The strong molecular weight dependence of the fraction of DNA recovered in the native form after a given time of pre-neutralization incubation at ethidium concentrations between the limiting values noted above allows larger DNAs to remain fully denatured upon neutralization while smaller DNAs in the same mixture are fully renatured. This permits the rapid fractionation of mixtures of closed duplex DNAs on the basis of molecular weight when a technique for the separation of denatured from fully base-paired DNA is applied to such mixtures. Such a separation has been demonstrated through the marked enrichment of plasmid cloning vector DNA containing cloned inserts in the fractions that remain denatured after neutralization of alkaline solutions of these DNAs containing ethidium bromide.

UI	MeSH Term	Description	Entries
D008970	Molecular Weight	The sum of the weight of all the atoms in a molecule.	Molecular Weights,Weight, Molecular,Weights, Molecular
D009691	Nucleic Acid Denaturation	Disruption of the secondary structure of nucleic acids by heat, extreme pH or chemical treatment. Double strand DNA is "melted" by dissociation of the non-covalent hydrogen bonds and hydrophobic interactions. Denatured DNA appears to be a single-stranded flexible structure. The effects of denaturation on RNA are similar though less pronounced and largely reversible.	DNA Denaturation,DNA Melting,RNA Denaturation,Acid Denaturation, Nucleic,Denaturation, DNA,Denaturation, Nucleic Acid,Denaturation, RNA,Nucleic Acid Denaturations
D009695	Nucleic Acid Renaturation	The reformation of all, or part of, the native conformation of a nucleic acid molecule after the molecule has undergone denaturation.	Acid Renaturation, Nucleic,Acid Renaturations, Nucleic,Nucleic Acid Renaturations,Renaturation, Nucleic Acid,Renaturations, Nucleic Acid
D002850	Chromatography, Gel	Chromatography on non-ionic gels without regard to the mechanism of solute discrimination.	Chromatography, Exclusion,Chromatography, Gel Permeation,Chromatography, Molecular Sieve,Gel Filtration,Gel Filtration Chromatography,Chromatography, Size Exclusion,Exclusion Chromatography,Gel Chromatography,Gel Permeation Chromatography,Molecular Sieve Chromatography,Chromatography, Gel Filtration,Exclusion Chromatography, Size,Filtration Chromatography, Gel,Filtration, Gel,Sieve Chromatography, Molecular,Size Exclusion Chromatography
D004269	DNA, Bacterial	Deoxyribonucleic acid that makes up the genetic material of bacteria.	Bacterial DNA
D004270	DNA, Circular	Any of the covalently closed DNA molecules found in bacteria, many viruses, mitochondria, plastids, and plasmids. Small, polydisperse circular DNA's have also been observed in a number of eukaryotic organisms and are suggested to have homology with chromosomal DNA and the capacity to be inserted into, and excised from, chromosomal DNA. It is a fragment of DNA formed by a process of looping out and deletion, containing a constant region of the mu heavy chain and the 3'-part of the mu switch region. Circular DNA is a normal product of rearrangement among gene segments encoding the variable regions of immunoglobulin light and heavy chains, as well as the T-cell receptor. (Riger et al., Glossary of Genetics, 5th ed & Segen, Dictionary of Modern Medicine, 1992)	Circular DNA,Circular DNAs,DNAs, Circular
D004279	DNA, Viral	Deoxyribonucleic acid that makes up the genetic material of viruses.	Viral DNA
D004926	Escherichia coli	A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc.	Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D004996	Ethidium	A trypanocidal agent and possible antiviral agent that is widely used in experimental cell biology and biochemistry. Ethidium has several experimentally useful properties including binding to nucleic acids, noncompetitive inhibition of nicotinic acetylcholine receptors, and fluorescence among others. It is most commonly used as the bromide.	Ethidium Bromide,Homidium Bromide,Novidium,Bromide, Ethidium,Bromide, Homidium
D006088	Gram-Negative Aerobic Bacteria	A large group of aerobic bacteria which show up as pink (negative) when treated by the gram-staining method. This is because the cell walls of gram-negative bacteria are low in peptidoglycan and thus have low affinity for violet stain and high affinity for the pink dye safranine.	Achromatiaceae,Achromatium,Achromobacteriaceae,Gram Negative Aerobic Bacteria