Deoxyribonucleic Acid
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Transcript of Deoxyribonucleic Acid
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Deoxyribonucleic Acid
(DNA)
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The double helix
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Nitrogenous Bases and Pentose Sugars
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Purine and Pyrimidine Structure
(1) Pyrimidines are planar (2) Purines are nearly planar(3) Numbering is different
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Numbering Is Different
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Bases Have Tautomeric Forms
Uracil
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Nucleosides vs. Nucleotides
Glycosidic bond
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Nucleotides formed by condensation reactions
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Monophosphates
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Deoxyribonucleotides
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Ribonucleotides
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Only RNA Is Hydrolyzed by Base
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Nucleoside Diphosphate and Triphosphate
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Dinucleotides and Polynucleotides
Ester bonds
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Watson-Crick Base Pairs
A=T
G=C
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Hoogsteen Base Pairs
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Other Base Pairs Are Possible
Homo Purines Hetero PurinesWatson-Crick,
Reverse Watson-Crick, Hoogsteen,
Reverse Hoogsteen, Wobble,
Reverse Wobble
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Base Pairing Can Result in Alternative DNA Structures
Triplex Tetraplex
Hairpin Loop Cruciform
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• Periodicity: A pair of strong vertical arcs (C & N atoms) indicate a very regular periodicity of 3.4 Å along the axis of the DNA fiber.
• Astbury suggested that bases were stacked on top of each other "like a pile of pennies".
• Helical nature: Cross pattern of electron density indicates DNA helix and angles show how tightly it is wound.
• Diameter: lateral scattering from electron dense P & O atoms.
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DNase can only cleave external bond demonstrating periodicity
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Watson and Crick Model (1953)• 2 long polynucleotide
chains coiled around a central axis
• Bases are 3.4 Å (0.34 nm) apart on inside of helix
• Bases flat & lie perpendicular to the axis
• Complete turn = 34 Å • 10 bases/turn• Diameter = 20 Å• Alternating major and
minor grooves
Hydrophobic
Hydrophilic
Complementarity
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Base Pairing Results from H-Bonds
Only A=T and GC yield 20 Å Diameter
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A:C base pair incompatibility
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Bases Are Flat
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Chains Are Antiparallel…
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Base Pairs and Groove Formation
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Base flipping can occur
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Helix Is Right-Handed
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Biologically Significant Form = B-DNA
Low Salt = Hydrated, 10.5 bp/turn
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A- DNA Exists Under High Salt Conditions
Side-view Top-view
Base pairs tilted, 23 Å, 11bp/turn
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Z-DNA Is a Left-Handed Helix
Zig-zag conformation, 18 Å, 12 bp/turn, no major groove
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Propeller Twist Results from Bond Rotation
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Reassociation Kinetics
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Denaturation of DNA Strands and the Hyperchromic Shift
• Denaturation (melting) is the breaking of H, but not covalent, bonds in DNA double helix duplex unwinds strands separate
• Viscosity decreases and bouyant density increases• Hyperchromic shift – uv absorption increases with
denaturation of duplex• Basis for melting curves because G-C pairs have three
H bonds but A-T pairs have only two H bonds• Duplexes with high G-C content have a higher melting
temperature because G-C pairs require a higher temperature for denaturation
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Molecular Hybridization
• Reassociation of denatured strands• Occurs because of complementary base pairing • Can form RNA-DNA Hybrids• Can detect sequence homology between species• Basis for in situ hybridization, Southern and
Northern blotting, and PCR
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Hybridization
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Reassociation Kinetics• Derive information about the complexity of
a genome• To study reassociation, genome must first
be fragmented (e.g. by shear forces)• Next, DNA is heat-denatured• Finally, temperature is slowly lowered and
rate of strand reassociation (hybridization) is monitored
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• Initially there is a mixture of unique DNA sequence fragments so hybridization occurs slowly. As this pool shrinks, hybridization occurs more quickly
• C0t1/2 = half-reaction time or the point where one half of the DNA is present as ds fragments and half is present as ss fragments
• If all pairs of ssDNA hybrids contain unique sequences and all are about the same size, C0t1/2 is directly proportional to the complexity of the DNA
• Complexity = X represents the length in nucleotide pairs of all unique DNA fragments laid end to end
• Assuming that the DNA represents the entire genome and all sequences are different from each other, then X = the size of the haploid genome
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The Tm
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The Hyperchromic Shift (Melting Curve Profile)
Tm = temperature at which 50% of DNA is denatured
Maximum denaturation = 100% single stranded
Double stranded
50% double, 50% single stranded
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High G-C Content Results in a Genome of Greater Bouyant Density
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Ideal C0t Curve
100% ssDNA
100% dsDNA
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Larger genomes take longer to reassociate because there are more DNA
fragments to hybridize
Largest genomeSmallest genome
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C0t1/2 Is Directly Proportional to Genome Size
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Genomes are composed of unique, moderately repetitive and highly repetitive
sequences
Highly repetitive DNA
Moderately repetitive DNA
10-4 10-2 100 102 104
Frac
tion
rem
aini
ngsi
ngle
-str
ande
d (C
/C0)
Unique DNA sequences
0
100
C0t (moles x sec/L)
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More complex genomes contain more classes of DNA sequences
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G-C Content Increases Tm