Explain the reasoning to question A. 8-9 In the classic paper that demonstrated

Question

Explain the reasoning to question A. 8-9 In the classic paper that demonstrated the semi-con servative replication of DNA. Meselson arid SahI began by showing tisai DNA Itself will Form a band when subjected to equilibrium sedimentation. Ibey mixed randomly 1mg mented E. coU DNA with a solution o?CsCl so that the tinal solution had a density of l.7 gimI. As shmvn in Figure QB?2. with Increasing length of centrifugation ai 70.000 times gravity, the DNA. which was initially dispersed throughout the tentriftige tube, became concentrated over Lime into a discrete band in usc middle A. Describe wha Is happening with time and explain why the I)NA lorms a discrete band. Figure OS-2 Ultraviolet absOrption photographs showing successive stages in the banding of L co )NA (Problem S-9). DNA, tiIch absorbs Uy hght. shows up as dar* regiam in the photographs. The bottom of the centrifuge tube is on the rigtil. iFrom M Meselsors and F.W. Stahl. P,Oc. NotlAcod, Sc? USA 44:671 68Z 1958. With permissson from National Academy of Sc.enc.s.I

Explanation / Answer

Three theories were proposed on how DNA might be replicated. They went by the names Conservative, Dispersive and Semi-conservative DNA replication. Semi-conservative DNA replication was the one proved to be true in DNA replication.

Semi-Conservative Replication is a process where each parental strand acts as a template for the synthesis of one new daughter strand. Therefore, in daughter molecules a newly synthesized strand is base paired to one of the original parental strands. thus the resulting daughter molecule will be a hybrid of one strand retained from the parent and one newly synthesized strand that base pairs.

To clarify this question the group used a way to isotopically label newly synthesized DNA. To do this they used two different isotopes of nitrogen (N). 14N most common form of nitrogen 15N less common form and has greater mass than 14N DNA made using 15N is about 1% denser than DNA made using 14N. These two forms can be separated by equilibrium density gradient centrifugation (also called isopycnic centrifugation).

Equilibrium density gradient centrifugation -
In this technique molecules mixed with a salt (CsCl2), dissolved in water, and centrifuged at very high speed. The salt molecules form a density gradient.Centrifuge tubes spinning in a centrifuge Prior to centrifugation the salt molecules are evenly distributed throughout the centrifuge tube. During centrifugation, the salt molecules are forced towards the bottom of the tube and a gradient of molecules is established. More of the salt is found at the bottom of the tube and less is at the top. Therefore, the density of the solution is
smaller at the top and greater at the bottom.
Any DNA molecules in this centrifuge tube will also be forced towards the bottom of the tube. As the DNA travels down the tube the density of the surrounding salt solution gradually increases. The DNA stops moving relative to the salt solution when their two densities are equal. This is because of a physical property called buoyancy.

Observations - 1. Prior to centrifugation, the DNA is evenly distributed throughout the centrifuge tubes. that means there are no bands.

2. Centrifugation begins and the salt and DNA begin sedimenting. the second tube has one band of DNA. which means that in this tube all of the DNA molecules have the same buoyant density and thus segregate at one point as one single band showing that they are all of the same bouyant density.

3. After prolonged centrifugation, the DNA has formed discrete bands reflecting its buoyant density. In the third observation, notice that the second tube has one band of DNA. This means that in this tube all of the DNA molecules have the same buoyant density. Also notice that the third tube has two bands. This means that the DNA in this tube is a mixture of DNAs with two different buoyant densities.

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