Experimental Proof for Semiconservative Replication of DNA in Bacteria

Semiconservative replication is the process by which DNA is replicated where each new DNA molecule consists of one original and one newly synthesized strand. This theory was proposed by Watson and Crick in 1953. However, the experimental proof for semiconservative replication was first demonstrated by Matthew Meselson and Franklin Stahl in 1958.

Experimental Procedure

1. Isotope Labeling

Meselson and Stahl used isotopes to differentiate between the original and newly synthesized strands of DNA. They grew E.coli bacteria in a medium containing a heavy isotope of nitrogen (15N) for several generations. This heavy isotope was incorporated into the bacterial DNA. The bacteria were then transferred to a medium containing a lighter isotope of nitrogen (14N) for a single generation. This allowed the newly synthesized DNA to be labeled with the lighter isotope.

2. Density Gradient Centrifugation

After one generation, the bacterial DNA was extracted and subjected to density gradient centrifugation. This technique separates molecules based on their density. The heavier DNA containing 15N settled at the bottom of the gradient while the lighter DNA containing 14N settled higher up in the gradient.

3. Analysis of DNA

The DNA samples collected from different parts of the gradient were analyzed for their density and the amount of DNA present. The DNA was analyzed using a technique called autoradiography which involves exposing the DNA to a photographic film to detect the presence of radioactive isotopes.

Results

Meselson and Stahl's experiment showed that the DNA isolated from the bacteria grown in the 14N medium was less dense than the DNA isolated from the bacteria grown in the 15N medium. This suggested that the newly synthesized DNA was lighter than the original DNA.

Furthermore, the DNA isolated from the bacteria grown in the 14N medium showed two distinct bands in the density gradient. One band was at the position of the original DNA (heavier band) while the other band was at a position between the original and newly synthesized DNA (intermediate band). This suggested that the newly synthesized DNA was a hybrid of the original and newly synthesized strands.

Conclusion

Meselson and Stahl's experiment provided the first experimental proof for semiconservative replication of DNA. The findings of their experiment showed that DNA replication occurs by a semiconservative mechanism where each daughter DNA molecule contains one original and one newly synthesized strand.