CsCl density gradient centrifugation
CsCl (cesium chloride) centrifugation is a method for separating DNA based on density. To separate DNA based on density, DNA is mixed with CsCl and centrifuged at very high speeds (e.g., 50,000 rpm) in an ultracentrifuge for many hours.
- Density gradient centrifugation as developed by Meselson and Stahl employed a process called equilibrium sedimentation or isopycnic centrifugation.
- Equilibrium sedimentation is the process by which particles in a solution reach a point where they reach their isopycnic position and stop moving.
- In isopycnic separation (buoyant or equilibrium separation) particles are separated solely on the basis of their density. Particle size only affects the rate at which particles move until their density is the same as the surrounding gradient medium.
Reason for stopping movement: The centrifugal force pushing the particles down equals the force of the solution pushing up, causing the particles to stop moving in the solution.
Meselson and Stahl could determine the density of different DNA molecules based on where they reached equilibrium and stopped moving, because the molecules would stop when their density matched the density of the surrounding solution.
- Density gradient centrifugation requires the use of a special type of centrifuge (Instrument that spins mixtures in a rotor to concentrate or separate materials) called an analytical ultracentrifuge or ultracentrifuge.
- CsCl solution with different DNA samples to be separated.
(To prevent the DNA molecules from breaking down in an acidic solution, the researchers maintained a constant level of pH six)
- The spinning causes sample solutions in tube or bottle shaped containers to experience a centrifugal force that pushes samples away from the center of the rotor toward the bottom of the tube.
- Centrifugal force causes components of a mixture to separate by size because larger components experience greater centrifugal force than smaller components.
- Thus, centrifugal force pushes the larger components of a mixture farther from the rotor and closer to the bottom of the tube.
- That means during centrifugation, density gradient forms in solution and the density of that solution gradually increases moving farther from the centrifuge rotor.
- The solution of the bottom of the tube became slightly denser than the solution at the top.
(In CsCl density gradient centrifugation, the force gradually pushed the cesium chloride salt particles toward the bottom of the tube, which resulted in a higher concentration of cesium chloride farther from the rotor. Cesium Chloride never formed a complete pellet at the bottom of the test tube because of its natural tendency to diffuse, or re-dissolve, back into solution)
The Meselson and Stahl Experiment used CsCl density gradient centrifugation for seperation of DNA based on density
- The researchers suggested tagging the original DNA with heavy elements that would not significantly alter the DNA´s chemical properties.
- While heavy elements would increase the weight of the DNA, they would not increase the size of the molecule, thereby only increasing the density of the original DNA.
- In developing their new density gradient centrifugation technique, Meselson and Stahl first chose a dense solution where the DNA molecules would separate.
- The solution needed to be dense enough for the DNA molecules to float before centrifugation.
Why they chosen CsCl density gradient for seperation?
The cesium chloride density had a density range greater than the difference in densities between the heavy and light DNA that Meselson and Stahl aimed to separate. Therefore, instead of centrifuging until the heavy DNA sank to the bottom of the container and the light DNA rose to the top of the container, Meselson and Stahl centrifuged the samples until the DNA suspended in separate parts of the solution and stopped moving at a point called equilibrium.
Experimental setup of Meselson and Stahl
Step 1: Preparation of 6 M CsCl solutions
Step 2: Addition of a mixture with14N containing DNA and 15N containing DNA
Step 3: Equilibrium density gradient centrifugation
When solutions are spun rapidly in an ultracentrifuge for 48-72hrs 250C, cesium chloride spontaneously formed a density gradient essential for Meselson and Stahl´s study of DNA.
If DNA is present in such a gradient, it will move to a position where the density of the CsCl solution is equal to its own density.
Step 4: observation the formation of concentrated DNA in the form of dark bands across the sample container
If a mixture of E. coli DNA containing the heavy isotope of nitrogen (15N) and E. coli DNA containing the normal light nitrogen isotope (14N) is subjected to CsCl equilibrium density-gradient centrifugation, the DNA molecules will separate into two “bands,” one consisting of “heavy” (15N-containing) DNA and the other of “light” (14N-containing) DNA.
The placement of the bands depended on the density of the DNA.
(Observation given here is that of control tube)
Step 5: Punch hole in the centrifuge tube and collect the sample separately.
Meselson Stahl experiment Result:
- The entire DNA isolated from cells after one generation of growth in medium containing 14N had a density halfway between the densities of “heavy” DNA and “light” DNA.
- This intermediate density is usually referred to as “hybrid” density.
- After two generations of growth in medium containing 14N, half of the DNA was of hybrid density and half was light.
The results of their experiment are consistent only with semiconservative replication, excluding both conservative and dispersive models of DNA synthesis.
These results are precisely those predicted by the Watson and Crick semiconservative mode of replication.