The Meselson-Stahl Experiment

The Meselson–Stahl experiment is personally one of my favorite biological experiments. It is an experiment by Matthew Meselson and Franklin Stahl in 1958 which supported Watson and Crick's hypothesis that DNA replication was semiconservative.

In the past, there were three models for how organisms might replicate their DNA: semi-conservative, conservative, and dispersive. Semi-conservative replication involves the two strands of DNA unwinding from each other, and each one acting as a template for synthesis of a new, complementary strand. Conservative DNA replication results in one molecule that consists of both original DNA strands (identical to the original DNA molecule) and another molecule that consists of two new strands (with exactly the same sequences as the original molecule). Dispersive DNA replication results in two DNA molecules that are mixtures, or “hybrids,” of parental and daughter DNA. In this model, each individual strand is a patchwork of original and new DNA.

The Meselson-Stahl experiment has been called "the most beautiful experiment in biology” and for very good reason. Meselson and Stahl decided the best way to label the parent DNA would be to change one of the atoms in the parent DNA molecule. Since nitrogen is found in the bases of each nucleotide, they used an isotope of nitrogen (this is an atom of the same element that has a different number of neutrons) to distinguish between parent and newly copied DNA. Since the isotope of nitrogen had an extra neutron in the nucleus, it was heavier.

Meselson and Stahl conducted their famous experiments on DNA replication using E. coli bacteria as a model system. They began by growing E. coli in a medium containing the "heavy" nitrogen. When grown on a medium containing heavy nitrogen, the bacteria took up the nitrogen and used it to synthesize new biological molecules, including DNA. After many generations growing in the “heavy” nitrogen medium, the nitrogenous bases of the bacteria's DNA were all labeled with the “heavy” isotope. Then, they switched the bacteria to a medium containing a "light" nitrogen isotope and allowed it to grow for several generations. DNA made after the switch would have to be made up of the “light” nitrogen, as this would have been the only nitrogen available for DNA synthesis.

Meselson and Stahl knew how often E. coli cells divided, so they were able to collect small samples in each generation and extract and purify the DNA. They then measured the density of the DNA (and, indirectly, its “heavy” and “light” nitrogen content) using density gradient centrifugation. This method separates molecules such as DNA into bands by spinning them at high speeds in the presence of another molecule, such as cesium chloride, that forms a density gradient from the top to the bottom of the spinning tube. Density gradient centrifugation allows very small differences—like those between the isotopes of nitrogen, to be detected

The results from the experiment are shown below:

Generation 0 is before the bacteria were placed into the medium containing “light” nitrogen and should only produce one band, because it is entirely “heavy” nitrogen.

Generation 1 shows an intermediate band in between the “light” and “heavy” bands. The intermediate band told Meselson and Stahl that the DNA molecules made in the first round of replication were a hybrid of light and heavy DNA. These results fit with the dispersive and semi-conservative models, but not with the conservative model, because the conservative model would have predicted two distinct bands.

Generation 2 produced two bands. One was in the same position as the intermediate band from the first generation, while the second was higher, meaning that the DNA was being replicated semi-conservatively. The pattern of two distinct bands—one at the position of a hybrid molecule and one at the position of a light molecule—is just what we'd expect for semi-conservative replication. We know that this is not dispersive replication because all the molecules should have sections of old and new DNA, making it impossible to get a "purely light" band. Generations 3 and 4 prove this further as the intermediate band begins to shrink.

By Donte