Question

In the classic paper that demonstrated the semi-conservative replication of DNA, Meselson and Stahl began by showing that DNA itself will form a band when subjected to equilibrium sedimentation. They mixed randomly fragmented E coli DNA with a solution of CsCl so that the final solution had a density of 1.71 g/ml. As shortn in Figure Q8-2, with increasing length of centrifugation at 70,000 times gravity, the DNA, which was initially dispersed throughout the centrifuge tube, became concentrated over time into a discrete band in the middle.

B. What is the buoyant density of the DNA? (The density of the solution at which DNA "floats" at equilibrium defines the "buoyant density" of the DNA.) C. Even if the DNA were centrifuged for twice as long-or even longer-the width of the band remains about what is shown at the bottom of Figure QB-2. why does the band not become even more compressed? Suggest some possible reasons to explain the thickness of the DNA band at equilibrlum.

Lupumb TUPILI the DNA forms a discrete band. hours centrifugal field 4.3 6.4 8.5 10.7 12.8 Figure Q8-2 Ultraviolet absorption photographs showing successive stages in the banding of E. coli DNA (Problem 8-9), DNA, which absorbs UV light, shows up as dark regions in the photographs. The bottom of the centrifuge tube is on the right. (From M. Meselson and F.W. Stahl, Proc. Natl Acad. Sci. U.S.A. 44:671-682, 1958. With permission from National Academy of Sciences.) 14.9 17.1 19.2 21.3 23.5 36.5 43.5 Type here to search

Answer 1

Buoyant density centrifugation or isopycnic centrifugation or equilibrium density-gradient centrifugation uses concept of buoyancy to separate molecules in solution by their differences in density.

Buoyant density of majority of DNA is 1.7g/cm³ which is equal to density of 6M CsCl solution. Buoyant density of DNA changes with its GC content( Guanine Cytosine content).

In Meselson and Stahl's method, centrifugal force pushing the particles down equals the force of the solution pushing up, causing particles to stop moving in the solution. The particles do not reach the bottom of the tube. So. the density of DNA molecules was determined based on where they reached equilibrium and stopped moving.

In their method, placement of bands depended on the density of DNA. Heavy and light DNA formed separate distinct bands with heavier DNA further down the density gradient.

The width of the band is inversely proportional to the centrifugal force.

The width of the band relates to the molecular weight and mean density of the molecules in that band. Since Mesleson and Stahl knew the approx molecular wt of their DNA molecules, they used bandwidth to calculate the density of the DNA molecle.

Since molecular weight and mean density of the E coli will have one particular value, so the width of the lower most band will become constant after a specific time period. Density of DNA in bottom most would have reached the mean density of E coli DNA molecule.That would be the heaviest DNA molecule in E coli. So, width of the band becomes constant corresponding to the heaviest molecule.