The Experimental Proof of DNA Replication

The Meselson–Stahl experiment provided the experimental proof that DNA replicates by the semiconservative method, as proposed by Watson and Crick.

Meselson and Stahl Experiment

In 1958, Matthew Meselson and Franklin Stahl designed a carefully controlled experiment using the bacterium Escherichia coli (E. coli) and stable isotopes of nitrogen to determine which model correctly explained DNA replication. Their observations provided conclusive evidence that DNA replicates by the semi-conservative mechanism.

Principle of the Experiment

• The Meselson and Stahl experiment was based on the principle that DNA molecules containing different isotopes of nitrogen possess different densities.
• The scientists used two stable isotopes of nitrogen are Heavy nitrogen (¹⁵N), which increases the density of DNA and Light nitrogen (¹⁴N), which produces DNA with a lower density.
• To separate DNA molecules according to their density, Meselson and Stahl used a technique known as caesium chloride (CsCl) density gradient centrifugation.
• During ultracentrifugation, a density gradient is formed within the caesium chloride solution, allowing DNA molecules of different densities to settle at different positions.
• Heavy DNA forms a band lower in the centrifuge tube, light DNA forms a band higher in the tube, and DNA containing both heavy and light nitrogen forms a band at an intermediate position.

Experimental Procedure

• Meselson and Stahl selected the bacterium Escherichia coli (E. coli) because it reproduces rapidly, making it ideal for studying DNA replication over several generations.
• Initially, the bacteria were grown for many generations in a nutrient medium containing only heavy nitrogen (¹⁵N). During this period, all newly synthesised DNA incorporated the heavy nitrogen isotope, so every DNA molecule in the bacterial cells became uniformly labelled with ¹⁵N. This produced DNA molecules that were denser than normal DNA.
• After ensuring that all bacterial DNA contained heavy nitrogen, the bacteria were carefully transferred into another nutrient medium that contained only light nitrogen (¹⁴N) instead of heavy nitrogen.
• From this point onward, every new DNA strand synthesised by the bacteria incorporated only the light nitrogen isotope.
• The bacterial cells were allowed to divide repeatedly in the ¹⁴N medium. Samples of bacterial DNA were collected after the completion of the first generation of replication and again after the second generation.
• Each DNA sample was then subjected to caesium chloride density gradient centrifugation so that DNA molecules of different densities could be separated and analysed.

Observations

Observation After the First Generation

• When the DNA extracted after one generation was examined using density gradient centrifugation, only one DNA band was observed.
• This DNA band was located exactly between the positions normally occupied by heavy DNA and light DNA, indicating that it possessed an intermediate density.
• The appearance of only one intermediate-density band demonstrated that every DNA molecule contained both heavy nitrogen and light nitrogen.
• In other words, each DNA molecule consisted of one original parental strand containing ¹⁵N and one newly synthesised strand containing ¹⁴N.
• This observation was entirely consistent with the predictions of the semi-conservative model of DNA replication.

Observation After the Second Generation

• The bacteria were then allowed to divide once more in the medium containing light nitrogen, and DNA was again isolated after the second generation.
• This time, two distinct DNA bands were observed during density gradient centrifugation. One band appeared at the intermediate position, representing hybrid DNA molecules containing one heavy strand and one light strand.
• The second band appeared at the position corresponding to light DNA, representing DNA molecules composed entirely of light nitrogen.
• The intermediate DNA band accounted for approximately half of the DNA molecules, while the light DNA band accounted for the remaining half.
• No completely heavy DNA band was observed because the original heavy DNA strands had already served as templates during replication and were no longer present together in the same DNA molecule.

Results

• The observations ruled out the conservative model because that model predicted one completely heavy DNA molecule and one completely light DNA molecule after the first generation.
• The observations also ruled out the dispersive model because dispersive replication would have produced only intermediate-density DNA after every generation rather than separate intermediate and light DNA bands.
• The results perfectly matched the predictions of the semi-conservative model, in which each daughter DNA molecule contains one parental strand and one newly synthesised strand.