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Buoyant Density and GC content, Cesium chloride centrifugation, Separation of Satellite DNA, separate supercoiled DNA from non-supercoiled molecules,EtBr-CsCl centrifugation
Buoyant Density of DNA
Density gradient centrifugation
• Density gradient centrifugation can be used to isolate DNA.
• The densities of DNAs are about the same as concentrated solutions of cesium chloride, CsCl (1.6 to 1.8 g/mL).
• Centrifugation of CsCl solutions at very high rotational speeds, where the centrifugal force becomes 105 times stronger than the force of gravity, causes the formation of a density gradient within the solution.
• This gradient is the result of a balance that is established between the sedimentation of the salt ions toward the bottom of the tube and their diffusion upward toward regions of lower concentration.
• If DNA is present in the centrifuged CsCl solution, it moves to a position of equilibrium in the gradient equivalent to its buoyant density.
• Caesium chloride is used because at a concentration of 1.6 to 1.8 g/mL it is similar to the density of DNA.
• For this reason, this technique is also called isopycnic centrifugation.
• The net movement of solute particles in an ultracentrifuge is the result of two processes: diffusion (from regions of higher concentration to regions of lower concentration) and sedimentation due to centrifugal force (in the direction away from the axis of rotation).
• In general, diffusion rates for molecules are inversely proportional to their molecular weight—larger molecules diffuse more slowly than smaller ones.
• On the other hand, sedimentation rates increase with increasing molecular weight. A macromolecular species that has reached its position of equilibrium in isopycnic centrifugation has formed a concentrated band of material.
• Cesium chloride centrifugation is an excellent means of removing RNA and proteins in the purification of DNA.
• The density of DNA is typically slightly greater than 1.7 g/cm3 (1.70+ 0.01), while the density of RNA is more than 1.8 g/cm3.
• Proteins have densities less than 1.3 g/cm3.• In CsCl solutions of appropriate density, the DNA
bands near the center of the tube, RNA pellets to the bottom, and the proteins float near the top.
• Single-stranded DNA is denser than double-helical DNA. The irregular structure of randomly coiled ssDNA allows the atoms to pack together through vander Waals interactions. These interactions compact the molecule into a smaller volume than that occupied by a hydrogen-bonded double helix.
• Density of DNA is dependent on relative G : C content. – G : C-rich DNA has a significantly higher density
than A :T-rich DNA.
• Furthermore, a linear relationship exists between the buoyant densities of DNA from different sources and their G : C content
• The density of DNA, (in g/mL), as a function of its G : C content is given by the equation
• where (GC) is the mole fraction of (G+ C) in the DNA.
• For every 10% increase in GC content, the density rises by 0.12 units.
Effect of methylation
• However, the presence of 5-methyl-cytosine serves to reduce the density slightly, thereby giving rise to an under-estimate of the GC content. In general, 1% methylation decreases the buoyant density by 1 mg cm–3 .
• Furthermore, single-stranded DNA is denser than double-stranded DNA of similar base composition by approximately 0.015 g cm–3 and under alkaline conditions the density is increased by 0.06 g cm–3 .
• This is due partly to the fact that DNA becomes single-stranded under these conditions and also partly because the deprotonated adenine and thymine residues are neutralized by binding caesium ions.
• Satellite DNA consists of highly repetitive DNA and is so called because repetitions of a short DNA sequence tend to produce a different frequency of the nucleotides and thus have a different density from bulk DNA - such that they form a second or 'satellite' band when genomic DNA is separated on a density gradient.
• For example, the fruit fly Drosophila virilis possesses a huge number of ACAAACT repeats that together add up to almost a quarter of the entire genome.
• The size of a satellite DNA ranges from 100 kb to over 1 Mb.
• ethidium bromide (EtBr) can be used to separate supercoiled DNA from non-supercoiled molecules.
• Ethidium bromide binds to DNA molecules by intercalating between adjacent base pairs, causing partial unwinding of the double helix.
• This unwinding results in a decrease in the buoyant density, by as much as 0.125 g/cm3 for linear DNA.
• However, supercoiled DNA, with no free ends, has very little freedom to unwind, and can only bind a limited amount of EtBr.
• The decrease in buoyant density of a supercoiled molecule is therefore much less, only about 0.085 g/cm3.
• As a consequence, supercoiled molecules form a band in an EtBr–CsCl gradient at a different position to linear and open-circular DNA