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Semi-Conservative Replication of DNA - Meselson & Stahl (1958)

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by Chelsea Payne on 4 March 2014

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Transcript of Semi-Conservative Replication of DNA - Meselson & Stahl (1958)

Materials and Methods
Experiment
-E. Coli grown at 36 degrees celcius in glucose
medium containing ammonium chloride as nitrogen source

-Microscopic cell counts and colony assays conducted to monitor growth

-N^15 E.coli grown for 14 generations in medium containing N^15H4Cl of a high purity
->Approximately 4x10^9 bacteria then removed and centrifuged

-Remaining cultures washed with N^14H4Cl and allowed to replicate once
-Replication stopped by the addition of adenine and uracil
->Sample of E. Coli then removed and centrifuged

-Excess adenine, guanine, uracil and cytosine then added to culture to allow another cycle of replication
-> Sample of E. Coli then removed and centrifuged
*Meselson and Stahl did not know the exact time for a complete cycle of E. Coli DNA replication, and this was determined by trial and error

Background
Previous studies had shown that DNA carries hereditary information and is capable of replicating

However, the exact mechanism of DNA replication was unknown, there were three possible theories

Meselson and Stahl carried out "the most beautiful experiment in biology" to determine the mechanism of replication
Discussion
Discussion
Heat denaturation

Salmon sperm retains its initial molecular weight

Escherichia coli dissociates into two subunits which are conserved during duplication
Meselson & Stahl
Results
N^15 forms a band closer to the outside,N^14 inside.
UV light absorbed by DNA
Outside is more dense, inside far end is less dense
"The heavier the species, the farther it goes outward"
-S.Carr
Buoyant Density Difference:0.014 gm/cm^3
discrete semi-conservative evidence vs. dispersive
banding at positions relative to density due to the density difference between N^14 and N^15.
Produces a "lighter" species (2 xN^14 strands) and a "hybrid" (N^14 strand + N^15 strand)
2 Generations: 50% "labeled" and 50% "unlabeled"
Source: S. Carr Bio2250 site, originally from iGenetics 3rd e., PJ Russell
Semi-Conservative Replication of DNA - Meselson & Stahl (1958)
"The Most Beautiful Experiment in Biology"

Matthew Meselson

(1930-)
Studied chemistry at the University of Chicago and graduated in 1951

Completed his Ph.D. with Linus Pauling

Accepted a position at Caltech working as an assistant professor

In 1960 he went on to teach molecular biology at Harvard
Franklin W. Stahl
(1929-)
Recieved a B.A from Harvard university in 1951

Completed his Ph.D. at the University of Rochester

Met Meselson at Woods Hall while attending a molecular biology class

In 1959 started teaching molecular biology at the University of Oregon

Explanation of Analytical Ultracentrifugation
-Experiment often presented as preparative centrifugation, which separates heavy sediment and less dense supernatant

-Meselson & Stahl actually used analytical centrifugation, which can separate materials in the "supernatant fraction" that vary only slightly in density

-Uses much higher centrifugal force

-Sample placed in centrifuge at right angle to axis of rotation and balanced by blank

-Rotor chamber cooled and in vacuum to reduce frictional heat at a high speed

-Progress of experiment monitored by UV light

-absorption of UV light by DNA produces a black band on photographic film, densitometer is the ‘real’ monitoring instrument, since it is much more sensitive to density differences than the film, or your eye looking at a print of the film

-Discrete changes in density at interface of layers bends light, which is analyzed using Schleiren Optical Imaging



The Three Theories
1)Semi-conservative: DNA strands break, each single strand is used as a template. Daughter DNA will contain one new and one old strand.

2)Conservative: Entire DNA molecule acts as a template. Daughter DNA will be comprised of newly synthesized DNA.

3)Dispersive: DNA backbone is broken every 10 nucleotides and unwinds. DNA is synthesized in short pieces, daughter strand contains mixture of old and new DNA.
Image adapted from original text
Source: http://disastergirrl.tumblr.com/post/22583259902/biochemistry-with-olivia-dna-replication
-Withdrawn samples chilled, centrifuged for 5 minutes, resuspended with NaCl and EDTA at pH 6, and lysed by sodium dodecyl

-Dodecyl lysate sulfate added to CsCl at pH 8.5 and buffered with tris(hydroxymethyl)aminomethane

-Centrifuged again at 25 degrees celcius for 20 hours so DNA could attain sedimentation equilibrium

-UV photographs taken during centrifugation analyzed by microdensitometer

-Density of partially labeled DNA determined by its relative position between fully labeled and non labeled species (with an estimated 2% margin of error)
The results are in accord with Watson-Crick Model

But they did not show:
DNA as polynucleotides chains
double helical structure of DNA
molecular weight can be calculated from a graph!
N^14 and N^15 labled DNA does not spontaneously band separately....
Conclusion
This would have never been possible if not for Watson & Crick!
This experiment is an extremely useful demonstration analytical centrifugation.

Meslson and Stahl solved the problem of DNA replication, proving that it will be semi-conservative in that a parent molecule will still pair with a new strand in subsequent generations.
It is important to remember that this experiment was a repeated series of cultures grown to various generation times rather than what is traditionally believed to be simply replicated in a single cuvette.
Another point that we should take home from this, is that we began to understand DNA replication from Meselson and Stahl's experiment

However, results can vary based only on the complexity of the organism itself.
References
Source: S. Carr Bio2250 site, originally from iGenetics 3rd e., PJ Russell
adapted from original text.
adapted from original text
http://erickbio.wordpress.com/2011/10/02/percobaan-meselson-stahl/
adapted from original text
adapted from original text
adapted from original text
http://www.dnaftb.org/20/bio-2.html
http://erickbio.wordpress.com/2011/10/02/percobaan-meselson-stahl/
http://disastergirrl.tumblr.com/post/22583259902/biochemistry-with-olivia-dna-replication
Campbell, Neil A. and Reece, Jane B. (2008) Biology (8th edition).
Stent (1971) "Molecular Genetics: an Introductory Narrative."
Source: S. Carr Biology 2250 site. Original source Stent (1971) "Molecular Genetics: an Introductory Narrative."
Personal Interest
Where are they now?
Stahl (84): retired 2001
http://www.molbio.uoregon.edu/facres/Stahl.html
Meselson (83):
https://www.mcb.harvard.edu/mcb/faculty/profile/matthew-s-meselson/

-Type of analytical centrifugation used is called density gradient ultracentrifugation

-Solvent in DNA experiments is CsCl

-Cs atoms are forced centrifugally towards bottom of tube

-Cs atoms also diffuse centripetally toward axis of rotation

-DNA migrates to the point where the density gradient is equal to its buoyant density (called "isopycnic point")
Videos and Links:
Matthew Meselson brief lecture:
DNA Learning Centre:
Density Gradient Ultracentrifugation
http://www.dnalc.org/view/15331-Proposed-models-of-DNA-replication-Matthew-Meselson-.html
DNA from the beginning:
http://www.dnaftb.org/20/av.html
Applications of this Finding
-Genomics research
->Human Genome Project
-> Gene mapping

-Molecular cloning

-Polymerase Chain Reaction (PCR)

Matthew Meselson is still at Harvard University, where he is currently the Thomas Dudley Cabot Professor of the Natural Sciences

Franklin Stahl still associates with the University of Oregon where he was a distinguished Emeritus Professor of Molecular Biology.
http://www.mun.ca/biology/scarr/iGen3_03-02.html
Carr, S.(Jan., 24th,2014)
Questions about Meselson Stahl
Experimental Paper
,Personal Communication
Wikipedia Searches: Frances Stahl and Matthew Meselson
Raylene Noftall, Chelsea Payne,
Paula Sacilatto,Danielle Sheaves
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