Even if three hypotheses were formulated, only the first two where actually looked into, the third one being eliminated as soon as some tests were made. The Watson and Crick model suggests semi-conservative replication, which must therefore be proven.

Conservative replication	Semi-conservative replication

The experiment

A culture of E. coli was grown in heavy nitrogen medium. During that phase, the bacteria incorporated heavy isotopes in its DNA. When enough of the bacteria are obtained, the culture is slowly centrifuged, and the medium containing the heavy isotopes discarded. A sample of the bacterial pellet is taken, and DNA prepared. If analysed on a gradient centrifugation, it is clear that the DNA is heavy: one band at high density is obtained. The rest of the pellet is resuspended in fresh medium with normal nitrogen and grown for a further 20 minutes before taking a sample for analysis. One band is obtained at a level intermediate between heavy and normal DNA. If a sample is taken a further 20 minutes afterwards, and the DNA analysed, two bands are obtained, one at the normal density for DNA, one at an intermediate level between heavy and normal.

Some questions

1) What has happened during the first 20 minutes after changing the medium?

2) Describe the DNA at this stage. Your description should fit the results of the experiment.

3) What is happening in the next 20 minutes?

4) Describe the DNA after 20 minutes.

5) Which model fits these results?

6) What results would you have had obtained for the other models?

Conclusions.

1) What has happened during the first 20 minutes after changing the medium?

E. coli is a fast growing organism whose cell cycle is well known. In the normal conditions of the experiment, the bacteria will have had time to go through one cycle of division in 20 minutes. After twenty minutes, there should be twice as much DNA and bacteria in the medium, but not more. During that time DNA has replicated just once.

2) Describe the DNA at this stage. Your description should fit the results of the experiment.

All the DNA has an intermediate weight: as DNA is two stranded it is logical to think one strand is heavy and one strand is normal weight. The two heavy strands of the original heavy DNA have separated, and DNA copied with normal light nucleotides. The new strands are light.

3) What is happening in the next 20 minutes?

In the next twenty minutes, the bacteria are going through another cycle of division, and DNA is replicated again. The medium is still normal.

4) Describe the DNA after 20 minutes.

We have now two kinds of DNA: one is intermediate, and fits the description above, and one is normal, therefore both strands are made up of normal nucleotides, taken out of the fresh medium. The original intermediate DNA have separated, and each strand copied. The heavy strand is copied and the resulting double stranded DNA is intermediate. The normal strand is copied and the resulting double stranded DNA is normal.

5) Which model fits these results?

The model that best fits this result is the semi-conservative replication .

6) What results would you have had obtained for the other models?

The model of conservative replication implies that there is conservation of the original template. It means that after twenty minutes we would have observed two bands (and not one), one being a heavy DNA band, and the other a normal DNA band. We would have never observed the intermediate DNA band.

The dispersive replication model suggests that the daughter DNA is a mixture of both parent and newly formed DNA. In the first generation DNA , we would have observed only intermediate DNA, but then in the second generation we would also observe one band, which will be slowly migrating toward a normal DNA as a greater percentage of normal nucleotide is incorporated.