Density Gradient Centrifugation

Meselson and Franklin used density gradient centrifugation to study the semi-conservative replication of DNA. This technique is based on the principle that molecules of different densities will settle at different positions in a gradient when subjected to centrifugal force.

Steps Involved in the Process

1. Growing bacteria in a medium containing a heavy isotope of nitrogen
The experiment involved growing E. coli bacteria in a medium containing a heavy isotope of nitrogen, 15N, for many generations. This resulted in all the bacterial DNA being labeled with 15N.

2. Transferring bacteria to a medium containing a lighter isotope of nitrogen
Next, the bacteria were transferred to a medium containing a lighter isotope of nitrogen, 14N, and allowed to grow for one generation.

3. Isolation of DNA
The bacterial cells were then harvested and their DNA was extracted.

4. Density gradient centrifugation
The DNA samples were subjected to density gradient centrifugation, which involved placing the DNA in a tube containing a gradient of cesium chloride. The tube was then spun at high speeds in a centrifuge, causing the DNA molecules to migrate to the position in the gradient that corresponded to their buoyant density.

5. Observations
The resulting DNA bands were observed under ultraviolet light. If DNA replication was conservative, the DNA band would have been a single band of intermediate density, whereas if it was semi-conservative, two bands would have been observed, one of intermediate density and one of lighter density.

6. Conclusion
Meselson and Franklin observed that the DNA bands were indeed two distinct bands, one of intermediate density and one of lighter density. This provided evidence for the semi-conservative replication of DNA.

Conclusion

Density gradient centrifugation was a crucial technique used by Meselson and Franklin to study the semi-conservative replication of DNA. This technique allowed them to separate the newly synthesized DNA from the parental DNA and provided evidence for the semi-conservative mode of DNA replication.