BIOL 3110 Lecture 3: Lecture 3 - Molecular Biology I
Monday, May 16, 2016
Topoisomerases, DNA Replication
BIOL 3110
Recap of last lecture:
Factors that contribute to the stability of dsDNA:
1. Hydrogen bonding between base pairs
•Two H bonds between A:T
•Three H bonds between G:C
2. Base stacking
•Bases are almost parallel to one
another and it turns with the helical turn
•B form is stable because the stacks are
parallel because the spaces are
hydrophobic so they try to shield one
another to avoid exposure to outside elements so they holds double helix together
•RNA doesn’t stack as well; the axis is tilted
-Why doesn’t RNA stack as well? This is because TNA forms in the A form and
because of the extra OH in the 5’ position so you cannot form the nice, neatly
stacked, B form helices.
•In the A form helices, there is a shift in the angle
of the helix so it can form many stacks, but it is
not stable
•RNA can only form A form helices and A form helices
has only some base stacking but the angle shifts so
rather than having parallel stacks, they are stacked
at angles relative to each other so they aren’t very
stable
*DNA duplex stability is due to sum of many weak
interactions
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Monday, May 16, 2016
DNA Complementarity and Hybridization:
-Complementarity of the DNA strands allows for the denature
and renature properties of dsDNA
-There is a natural tendency to find the partner and to form the H
bonds again and form the stacking again because that is the
most stable form for the DNA to be in
-Many molecular biology techniques are based on the concept of
hybridization
•ex. Can be used to compare genomes; If you have equal
molarity or excess of one over the other, they will form hybrids
depending on the commonality of the genomes
-What other parameters influence the stability of the DNA
duplex? If we have species A and species B, more or less
related, then first we denature the DNA using heat (make
dsDNA into ssDNA) and then hybridize them together.
•Because we have excess, the molecules start to form these
hybrids to the one strand on one species (depending on the similarity and the
bases) can form hybrids with the other species
•Now we can compare genomes
•The closer you are, the more likely that these sequences are conserved
Properties of DNA:
-Because the nts are ring structures, they have the ability to absorb UV
-We typically measure the absorbance at 260nm
•That is the average peak absorbance of the 5 nts
-Because of base stacking, dsDNA absorbs less UV so the same amount of dsDNA
and ssDNA will absorb different amounts
-Each nt type has characteristic UV-light absorption profile
-Absorption of UV by ssDNA and dsDNA is different = hyperchromicity
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Monday, May 16, 2016
-Transition from dsDNA to ssDNA during denaturation -> Hyperchromic Shift
-Midpoint of hyper chromic shift = Tm, Melting Temperature = 50% dsDNA & 50%
ssDNA
-You only get shift because they are perfectly complementary to each other
-NOTE: When we are doing molecular biology experiments, we want 100% annealing
-In PCR, we have primers that are complementary to the template strand, so you want
all of this to be 100% annealing
-We have determined that Tm is 50% annealing. If you want 100% annealing,
what temperature would you chose? You want a temperature that is a few degrees
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Document Summary
*dna duplex stability is due to sum of many weak interactions. Complementarity of the dna strands allows for the denature and renature properties of dsdna. There is a natural tendency to nd the partner and to form the h bonds again and form the stacking again because that is the most stable form for the dna to be in. Many molecular biology techniques are based on the concept of hybridization: ex. Can be used to compare genomes; if you have equal molarity or excess of one over the other, they will form hybrids depending on the commonality of the genomes. Because the nts are ring structures, they have the ability to absorb uv. We typically measure the absorbance at 260nm: that is the average peak absorbance of the 5 nts. Because of base stacking, dsdna absorbs less uv so the same amount of dsdna and ssdna will absorb different amounts. Each nt type has characteristic uv-light absorption pro le.
