Mohammad Ahmed Hotiana (17-10532)
BIOT 313 (Molecular Biology)

THE REPLICATION OF DNA IN ESCHERICHIA COLI
By Mathew Meselson & Franklin W Stahl
Biology Vol. 44, 1958

Introduction:
Deoxyribonucleic acid (DNA) molecules are responsible for the transfer of hereditary information from one generation to another. DNA replication is the process by which DNA molecules replicate to produce identical copies that are to be transferred to the daughter cells.
Because of its long history as a sample organism for many biological experiments and its ability to replicate at a fast rate under normal conditions, researches used Escherichia Coli bacteria for this experiment.

Hypothesis:
After replication of a DNA molecule, each daughter molecule contains one parental strand and one newly synthesized strained; therefore undergoing replication in accordance to the semiconservative mechanism.

Technique:
By introducing N15 isotope of nitrogen (that has a higher density as compared to that of the highly abundant N14 isotope), density-gradient centrifugation can be used to analyze the DNA samples of different generations. Therefore, conforming the mechanism of DNA replication.

Experimentation:
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E.Coli B bacteria was grown in a medium containing ammonium chloride as the only source of nitrogen.

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Preparation of bacteria labelled with N15:
Growing washed cells (to a titer of 2 x 108 /ml) in a medium containing 100 ug/ml of
N15H4Cl (of 96.5 percent isotopic purity) for 14 generations.

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Abrupt change to N14:
Addition of ten-times N14H4Cl.

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4 x 109 bacteria were isolated from the culture before the change to N14 and at intervals afterwards for numerous generations.

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Preservation of DNA molecules and cell lyses:
Samples obtained were immediately chilled and centrifuged in the cold for 5 minutes at
1,800 X g. After placing in 0.40 ml of a cold solution [0.01 M in NaCl and 0.01 M in ethylenediaminetetra-acetate (EDTA)] at pH 6, the cells were lysed by the addition of
0.10 ml of 15 per cent sodium dodecyl sulfate and stored in the cold.
(Note: These conditions were introduced to make sure the cell is destroyed while preserving the molecules under concentration, DNA. EDTA was used as a metal scavenger, making sure no available metal takes part in any reaction)

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Density-gradient centrifugation:
(As a result of this centrifugation a density gradient is established and molecules settle at different positions in accordance to their densities)

0.010 ml of the dodecyl sulfate lysate was added to 0.70 ml of CsCl solution [buffered at pH 8.5 with 0.01 M tris aminomethane]. The density of the resulting solution was 1.71 gmcm-3. This was centrifuged at 44,770 rpm for 20 hours, until the DNA had reached sedimentation equilibrium.
Isolated bands of DNA were observed at a region of density 1.71 gmcm-3.
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Microdensitometer was used to obtain ultraviolet absorption photographs for each centrifugation. Result:
For the F1 (one generation time) generation, the band observed was in-between the bands for
N14 and N15. This shows that only hybrid molecules were present in this case.
For the F2 (two generation times) generation, the two equal bands observed were in accordance to the N14 (unlabeled) DNA and the hybrid DNA molecules. This shows that in this case there was an equal proportion of unlabeled and hybrid DNA, while the original parental N15 DNA was not present.

Conclusion:
In the light of the results obtained by this experiment and the DNA structure presented by
Watson and Crick, it can be concluded that each daughter DNA molecule contains one parental and one newly synthesized strand.
Therefore, DNA replication is achieved via semiconservative replication mechanism.

Heat denaturation:
Meselson and Stahl also compared the DNA of E.coli with salmon-sperm DNA in respect to their behavior upon heat denaturation.
It was observed that E.Coli DNA breaks down easily as compared to salmon-sperm DNA.
According to the researches this could either be due to more stable bonds within the salmonsperm DNA molecules, or it could be because of a difference in the DNA sub-units in case of the two organisms. It is worth noting that the later assumption would challenge the universal model of DNA presented by Watson and Crick.