Biotechnology and Bioinformatics (1+1)

1.4.7.1. Meselson-Stahl experiment

The semi-conservative theory can be confirmed by making use of the fact that DNA is made up of nitrogen bases. Nitrogen has an isotope N15 (N14 is the most common isotope) called heavy nitrogen. The experiment that confirms the predictions of the semi-conservative theory makes use of this isotope.

1. Bacterial (E. coli) DNA is placed in a media containing heavy nitrogen (N15), which binds to the DNA, making it identifiable.

2. This DNA is then placed in a media with the presence of N14 and left to replicate only once. The new bases will contain nitrogen 14 while the originals will contain N15

3. The DNA is placed in test tubes containing caesium chloride (heavy compound) and centrifuged at 40,000 rpm.

4. The cesium chloride molecules sink to the bottom of the test tubes creating a density gradient. The DNA molecules will position at their corresponding level of density (taking into account that N15 is more dense than N14)

5. These test tubes are observed under uv-rays. DNA appears as a fine layer in the test tubes at different heights according to their density

According to the semi-conservative theory, after one replication of DNA, we should obtain 2 hybrid (part N14 part N15) molecules from each original strand of DNA. This would appear as a single line in the test tube. This result would be the same for the dispersive theory. On the other hand, according to the conservative theory, we should obtain one original DNA strand and a completely new one i.e. two fine lines in the test tube placed separately one from the other. Up to this point, either the semi-conservative or the dispersive theories could be truthful, as experimental evidence confirmed that only one line appeared after one replication. In order to conclude between those two, DNA had to be left to replicate again, still in a media containing N14.

In the dispersive theory, after 2 divisions we should obtain a single line, but further up in the test tube, as the DNA molecules become less dense as N14 becomes more abundant in the molecule. According to the semi-conservative theory, 2 hybrid molecules and 2 fully N14 molecules should be produced, so two fine lines at different heights in the test tubes should be observed.

Experimental evidence confirmed that two lines were observed providing evidence for the semi-conservative replication theory.