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Lecture 2

BIOB11H3 Lecture Notes - Lecture 2: In Situ Hybridization, Hydrogen Bond, Polynucleotide


Department
Biological Sciences
Course Code
BIOB11H3
Professor
Dan Riggs
Lecture
2

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1
Lecture 2
Genome organization and
molecular evolution
SPs: Fig 10-10, 15, 17, 19, 22, 23
Vocabulary: Absorption spectrum/Denaturation/Hybridization
Renaturation or Reannealing/
in situ
hybridization
Centromere/Telomere /Mutation/Unequal Crossing over
Duplication/deletion/Transposition/Gene family
DNA: double stranded, antiparallel helix
Complementary base pairing
occurs by hydrogen bonding; A-T
and C-G.!
Fig 10-10
5’ 3’
3’ 5’
Early work on DNA: Physiochemical approaches
1. DNA is a polynucleotide chain
2. Estimates made of genome size
3. Values were ‘tiny yet enormous’
Humans: 3.5 picograms of DNA/haploid
genome = 3.2 billion base pairs
5. DNA absorbs light in ultraviolet range
4. Base pairing rules established by analyzing
base composition
6. Renaturation experiments defined complexity
Chargaff: (1950s) base composition
differs between organisms
[A] = [T] [G] = [C] [A] + [T] [C] + [G]
Fig 10-10C
A-T: 2 hydrogen bonds
C-G: 3 hydrogen bonds

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2
Absorbance measured by a spectrophotometer
0 25 50 75 100
Lamp
Monochromator
Cuvette Holder
Slide rod
Standard
Sample
Phototube Meter
DNA absorption maximum !260nm
Absorbance can be used to
determine DNA concentration
Ring structures of DNA absorb in the ultraviolet range
Absorbance increases about 1.5X if DNA is denatured.
Thus, changes in absorbance reveal % DS vs. %SS
Fig 10-15
50% dsDNA
‘Tm’
Why? Consider the “satellite dish” analogy
dsDNA: more
rigid, nitrogenous
bases limited in #
of positions they
can occupy.
ssDNA; flexible;
nitrogenous bases
can freely rotate
about glycosidic
bond
Complexity: a measure of the number of unique
(vs. repetitive) sequences that exist in a genome.
DNA reannealing (& later use of hybridization
techniques) is useful for determining aspects of
complexity.
5’ A-T-T-C-A-T-G-C-A-T-T-A-G-G-C-T-A-T 3’
3’ T-A-A-G-T-A-C-G-T-A-A-T-C-C-G-A-T-A 5’
the nitrogenous bases absorb UV light
ds becomes single
stranded
ds dna absorbs less than single stranded dna bc:
in single stranded dna the bases can face the light
since they can rotate freely and absorb more light
can rotate b/c no H bonds
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