BIOL340 - DNA REPLICATION EXAM QUESTIONS AND
ANSWERS WITH COMPLETE SOLUTIONS VERIFIED
Three alternative schemes for DNA replication
•Dispersive replication
- dispersive: original molecule is broken up and the new one is a mixture of a bunch of
different pieces both new and old, dispersed between the two.
•Conservative replication
-conservative: original molecule is still intact and you have just copied it
•Semiconservative replication
DNA replication is semiconservative
Meselson-Stahl experiment
•Used heavy 15N and light 14N isotopes of nitrogen to distinguish parental from newly
synthesised DNA
•Grew bacteria in 15N to incorporate into bases. Washed and placed in 14N.
- grew in E. coli. DNA is made from amino acids, bacteria is forced to make all of its
amino acids using these 15N nitrogen molecules (by putting in 15N ammonium
chloride), these amino acids then get incorporated into the DNA. incorporation of 15N or
14N depending on the media the bacteria were grown in.
•Grew and removed samples at intervals over several generations
•Extracted DNA and analysed by CsCl density centrifugation
- you end up with a density gradient with the most dense at the bottom.
,- specific type of density gradient centrifugation, called isopycnic [WHAT] centrifugation
that used a solution of cesium chloride to separate DNA molecules based on density
alone [google]
- there are two opposing forces, one is the G force pushing it down and the other is
natural buoyency within the gradient driving it up and it will equilibrate at the level where
the G force and buoyency are equal (i.e. at its ideal density)
15N is heavier so the point at which these two molecules will come to rest will be
different beacuse they have different densities.
you can see this by UV absorption - they shine UV light at the tube and they put a
photographic plate behind it and whereby the DNA is will absorb the UV light and create
a shadow = called UV shadowing
- 15N is not radioactive.
IMAGE (DNA replication, S7) - controls
- E.coli grows slower in ammonium chloride than it does it rich media.
- proves they were capable of resolving DNA labelled with 14N from 15N on the basis of
density.
Meselson-Stahl
•Which replication scheme does the data support?
•Why does it exclude the others?
from 0 to 1.0 generations the DNA is less dense.
by 1.0 generations with conservative you would see two bands because the the copy
would be produced in 14N media. with dispersive you would expect 1 band. with semi-
conservative you would also see one band.
, in 1.9 there are two bands which supports the semi-conservative. but the definitive proof
is where they mix the two together and we get three bands. if it was dispersive, you
would only see one band because it would go from containing 15N 100% to 50 to 25 so
the band would move left but only single band would be present which would be of
intermediate size and would just continue to move left.
the more generations that pass the more 14N copies you have but you always still have
the 15N strand so even as the 14N band gets darker you can still see the 15N band.
image (S8)
DNA replication
Three activities associated with replication of duplex DNA
•Initiation
- recognition of an origin
- separation and stabilization of parental strands
•Elongation
- assembly of the replisome at the replication fork synthesis of the daughter strands
•Termination
- joining at the ends
separation of the chromosome
refers to all the components which get recruited and recognise an origin of replication
where replication starts (specific sequence)
DNA replication in E.coli
(highly conserved for both prokaryotes and eukaryotes)
•Prokaryotic replication is better understood than eukaryotic
ANSWERS WITH COMPLETE SOLUTIONS VERIFIED
Three alternative schemes for DNA replication
•Dispersive replication
- dispersive: original molecule is broken up and the new one is a mixture of a bunch of
different pieces both new and old, dispersed between the two.
•Conservative replication
-conservative: original molecule is still intact and you have just copied it
•Semiconservative replication
DNA replication is semiconservative
Meselson-Stahl experiment
•Used heavy 15N and light 14N isotopes of nitrogen to distinguish parental from newly
synthesised DNA
•Grew bacteria in 15N to incorporate into bases. Washed and placed in 14N.
- grew in E. coli. DNA is made from amino acids, bacteria is forced to make all of its
amino acids using these 15N nitrogen molecules (by putting in 15N ammonium
chloride), these amino acids then get incorporated into the DNA. incorporation of 15N or
14N depending on the media the bacteria were grown in.
•Grew and removed samples at intervals over several generations
•Extracted DNA and analysed by CsCl density centrifugation
- you end up with a density gradient with the most dense at the bottom.
,- specific type of density gradient centrifugation, called isopycnic [WHAT] centrifugation
that used a solution of cesium chloride to separate DNA molecules based on density
alone [google]
- there are two opposing forces, one is the G force pushing it down and the other is
natural buoyency within the gradient driving it up and it will equilibrate at the level where
the G force and buoyency are equal (i.e. at its ideal density)
15N is heavier so the point at which these two molecules will come to rest will be
different beacuse they have different densities.
you can see this by UV absorption - they shine UV light at the tube and they put a
photographic plate behind it and whereby the DNA is will absorb the UV light and create
a shadow = called UV shadowing
- 15N is not radioactive.
IMAGE (DNA replication, S7) - controls
- E.coli grows slower in ammonium chloride than it does it rich media.
- proves they were capable of resolving DNA labelled with 14N from 15N on the basis of
density.
Meselson-Stahl
•Which replication scheme does the data support?
•Why does it exclude the others?
from 0 to 1.0 generations the DNA is less dense.
by 1.0 generations with conservative you would see two bands because the the copy
would be produced in 14N media. with dispersive you would expect 1 band. with semi-
conservative you would also see one band.
, in 1.9 there are two bands which supports the semi-conservative. but the definitive proof
is where they mix the two together and we get three bands. if it was dispersive, you
would only see one band because it would go from containing 15N 100% to 50 to 25 so
the band would move left but only single band would be present which would be of
intermediate size and would just continue to move left.
the more generations that pass the more 14N copies you have but you always still have
the 15N strand so even as the 14N band gets darker you can still see the 15N band.
image (S8)
DNA replication
Three activities associated with replication of duplex DNA
•Initiation
- recognition of an origin
- separation and stabilization of parental strands
•Elongation
- assembly of the replisome at the replication fork synthesis of the daughter strands
•Termination
- joining at the ends
separation of the chromosome
refers to all the components which get recruited and recognise an origin of replication
where replication starts (specific sequence)
DNA replication in E.coli
(highly conserved for both prokaryotes and eukaryotes)
•Prokaryotic replication is better understood than eukaryotic
