DNA

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DNA is the genetic material Study 1-Griffiths
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Study 2-Avery, MacLeod and McCarty
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DNA is the genetic material Study 3 Hershey and
Chase
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DNA structure
The genetic material of all free living
organisms (some viruses have RNA
genomes) Chemical Composition Pentose Sugar
Deoxyribose (ribose in RNA) Phosphate -
phosphodiester (strong acid)
Nitrogenous Bases (G) guanine (C) cytosine
(A) adenine (T) thymine At
physiological pH, every residue carries a net
negative charge
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Nomenclature
Base Sugar Nucleoside Deoxy-guanosine (DNA)
Deoxy-adenosine Deoxy-cytidine Deoxy-t
hymidine Nucleoside Phosphate(s)
Nucleotides e.g. nucleotide ATP (Adenosine
TriphosPhate) e.g. nucleotide GDP (Guanosine
DiphosPhate)
adenosine Triphosphate
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Chemical Structure of DNA and RNA
Figure 4.1
The Cs in sugar are named 1-5
Glycosidic bond
1
4
2
Nucleotide
Nucleoside
DNA
RNA
Sequence of the bases is called the primary
structure
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Bases of DNA (and RNA)
Purines
Forms Glycosidic bond with 1 C
Pyrimidines
DNA only
RNA only
You should be able to draw a short strand of RNA
or DNA, and the 5 bases
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Base-PairingAn explanation of how DNA could be
accurately replicated in the cell
AT 2 H-Bonds GC 3 H-Bonds
Figure 4.10
You should know how these bases interact with
each other
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Chargaffs Rules for all double-stranded (ds)
DNAs

• A T and G C
• A G T C

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Space Filling Model of B-DNA
Secondary structure of DNA The double
helix Watson and Cricks model 1953- based on
X-ray diffraction
helix is right-hand
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Primary Structure of DNA 5- 3 sequence of
bases
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Secondary Structure of DNA Right-hand double
helix highly stable structure maintained
largely through hydrogen bonding between
complementary bases and base stacking (van der
Waals) forces between adjacent bases
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Grooves in dsDNA
minor
major
B A Forms of DNA have two non-equivalent
grooves - major minor grooves - grooves are
openings between successive turns of the
sugar-phosphate backbone - BASE-PAIRS are exposed
to solvent at the grooves
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Grooves
- each Watson Crick base pair, GC, AT, CG,
TA has a major groove side and a minor groove
side. - on either side (especially the major)
there is potential for the base pairs to H-bond
with external groups. Like water or proteins. -
functional groups of base pairs that can be
accessed from grooves (-NH2, CO, ring N) serve
as contact points for proteins searching for
specific sequences on the DNA -hydrophobic and
van der Waals forces also act between adjacent
bases to contribute to stability
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Properties of dsDNA
- two strands are held together by hydrogen
bonds (between complementary base pairs)
base-pair stacking (between adjacent pairs) or
van der Waals interactions. - strands run
anti-parallel 5 ? 3 3 ? 5 - both the
formation disruption of DNA double helices are
highly cooperative
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Types of DNA Double Helices
There are THREE types of geometries for DNA
double helices Right Handed Helices B-Form
found in the cell Watson Crick Model A-Form
Shorter, Broader helix dsRNA and RNA-DNA hybrids
form A-type helices Left Handed Helix Z-Form
can be formed with particular sequences under
special circumstances with alternating Purines
Pyrimidines)
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A B Z
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Figure 4.14 B- and A-DNA
B-DNA - 3.4 nm/turn - 23.5 Å wide - 10 bp/turn
A-DNA - 2.8 nm/turn - 25.5 Å wide - 11 bp/turn -
Base-pairs tilted about 19 degrees from axis of
the helix.
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Z-DNA
- 4.5 nm/turn - 18.4 Å wide - 12 bp/turn -
base-pairs tilted about 9 degrees from axis of
the helix
Major groove
Minor groove
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Using Spectroscopy to analyze DNA(more
information on pages 202-204)
DNA absorbs UV light with a major peak at 260 nm
This absorption is useful because it varies with
the structure of DNA (RNA) i.e. extinction
coefficient depends on the structure and relates
directly to how much light that structure absorbs
Optical Density
Wave Length
260
Can relate structure (ie. Extinction coefficient)
and absorption with concentration
ssDNA Higher extinction coefficient
dsDNA Low extinction coefficient
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• Hyperchromic shift
• Nucleic acids absorb light at 260 nm
• When two strands come together they quench the
  absorbance
• When they denature, the quenching disappears and
  the absorbance rises 30-40
• Shows the strands hold fast until the Tm and then
  rapidly let go (i.e. co-operativity)

HYPERCHROMIC SHIFT
Optical Density (_at_260 nm)
Temperature (C)
Tm Melting Temp. Point at which strands are
half denatured
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Monitoring the melting temperature of dsDNA
Melting (denaturation) temperature depends on
four major factors - GC content (and therefore
AT content) (gtGC Tm lt GC Tm) - salt
conc. (gtsalt Tm ltsalt Tm) - sequence
length - gaps (base pairing mismatches in the
annealed strands)
GC rich
Temp
Temp
AT rich
GC rich
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• GC content affects Tm since higher GC ratio
  means the DNA is joined by 3 hydrogen bonds
  versus 2 for AT (note that The GC content of
  the DNA from different organisms can vary from
  22 - 73) the length of DNA (i.e. the total
  number of H bonds) will obviously affect Tm
• Lowering the salt concentrations removes ions
  that shield negative charges on DNA (i.e. the
  phosphates in the sugar-phosphate backbone) from
  one another this will promote the denaturation
  of DNA at relatively low temp (i.e. will lower
  the Tm) increasing the salt concentrations can
  do the opposite (raises the Tm)
• Organic solvents such as DMSO (dimethyl
  sulfoxide) and formamide disrupt H bonding
  between DNA strands i.e. they compete for
  H-bonding between the bases, thereby lowering the
  Tm
• pH changes can also disrupt H bonding