deoxyribonucleic acid (dna). the double helix nitrogenous bases and pentose sugars
TRANSCRIPT
Deoxyribonucleic Acid
(DNA)
The double helix
Nitrogenous Bases and Pentose Sugars
Purine and Pyrimidine Structure
(1) Pyrimidines are planar
(2) Purines are nearly planar
(3) Numbering is different
Numbering Is Different
Bases Have Tautomeric Forms
Uracil
Nucleosides vs. Nucleotides
Glycosidic bond
Nucleotides formed by condensation reactions
Monophosphates
Deoxyribonucleotides
Ribonucleotides
Only RNA Is Hydrolyzed by Base
Nucleoside Diphosphate and Triphosphate
Dinucleotides and Polynucleotides
Ester bonds
Watson-Crick Base Pairs
A=T
G=C
Hoogsteen Base Pairs
Other Base Pairs Are Possible
Homo Purines Hetero PurinesWatson-Crick,
Reverse Watson-Crick, Hoogsteen,
Reverse Hoogsteen, Wobble,
Reverse Wobble
Base Pairing Can Result in Alternative DNA Structures
Triplex Tetraplex
Hairpin Loop Cruciform
• 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.
DNase can only cleave external bond demonstrating periodicity
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
Base Pairing Results from H-Bonds
Only A=T and GC yield 20 Å Diameter
A:C base pair incompatibility
Bases Are Flat
Chains Are Antiparallel…
Base Pairs and Groove Formation
Base flipping can occur
Helix Is Right-Handed
Biologically Significant Form = B-DNA
Low Salt = Hydrated, 10.5 bp/turn
A- DNA Exists Under High Salt Conditions
Side-view Top-view
Base pairs tilted, 23 Å, 11bp/turn
Z-DNA Is a Left-Handed Helix
Zig-zag conformation, 18 Å, 12 bp/turn, no major groove
Propeller Twist Results from Bond Rotation
Reassociation Kinetics
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
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
Hybridization
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
• 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
The Tm
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
High G-C Content Results in a Genome of Greater Bouyant Density
Ideal C0t Curve
100% ssDNA
100% dsDNA
Larger genomes take longer to reassociate because there are more DNA
fragments to hybridize
Largest genomeSmallest genome
C0t1/2 Is Directly Proportional to Genome Size
Genomes are composed of unique, moderately repetitive and highly repetitive
sequences
Highly repetitive DNA
Moderately repetitive DNA
10-4 10-2 100 102 104
Fra
ctio
n r
emai
nin
gsi
ngl
e-st
ran
ded
(C
/C0)
Unique DNA sequences
0
100
C0t (moles x sec/L)
More complex genomes contain more classes of DNA sequences
G-C Content Increases Tm