Download LECT14 DNA

DNA
Deoxyribonucleic Acid
PROPERTIES OF DNA
PRIMARY SEQUENCE
A. Structural Representations
B. Historical Perspective
C. X-Ray evidence
D. Palindrome
E. Restriction nucleases
F. DNA Sequencing
G. Polymerase Chain Reaction
BASICS
3’ end
5’ end
pApGpCpT
A
G C
T
3’ end
P
5’ end
P
5’
P
P
OH
Historical Perspective
• Miescher and Civil War Bandages (1860’s)
• Avery bacterial DNA alters phenotype of
pathogenic pneumococci (1940’s)
• Hershey and Chase 32P, not 35S –labeled T2
bacterial phage entered the bacteria
• Pauling showed helix in proteins and cross
pattern on X-ray
• Chargaff showed that A = T and C = G
• Watson and Crick solve structure using X-ray
diffraction and model building
Rosalind Franklin’s X-Ray Photo Showing Regular
Repeating Patterns in DNA Structure
Cross Pattern
indicates Helix
Helical rise
3.4 nm
Layer line spacing
1/10 from center to spot
(10 base pairs per repeat)
Wet DNA Fibers
Deductions from X-Ray and Other
Data
• Cross pattern indicates helix
• Density data indicates two DNA strands
• Two stand helix stabilized by H-bonding if
bases were paired in a particular way:
– A-T and G-C, i.e., purine to pyrimidine to allow
regular diameter (not possible with purine to
purine or pyrimidine to pyrimidine)
CONFORMATION OF SUGAR-PHOSPHATE
Nucleotides in DNA have 7 torsion angles that
govern orientation of nucleotide chain.
O
O
HN
H2N
interaction
N
N
N
N
N
HOCH2
O
HOCH2
O
N
NH2
Favorable
Unfavorable
HO OH
Z-DNA
syn-Guanosine
NH
HO OH
anti-Guanosine
TYPES OF DNA
1. 3 types: A, B, and Z
2. Not in equilibrium
3. Transition depends on humidity, temperature
and DNA binding proteins
B-DNA
B-DNA (Watson-Crick) 90% humidity
1. Two Antiparallel polynucleotide strands
2. Sugar phosphates on periphery
(Minimize charge repulsion)
3. Helix approximately 20 Angstroms in diameter
4. 10.5 base pairs per turn, ~36 degrees per base pair
5. Bases flat, perpendicular to axis
6. Major and minor grooves readily apparent
Major
Minor
A DNA:
What distinguishes A DNA from B DNA?
A DNA is wider and flatter: 11 base-pairs per turn instead
of 10.5. The helix diameter is 26 angstroms instead
of 20. The major groove is narrow and subdued.
Is base-pairing the same?
Yes. But the bases join around the axis and not through
the axis and are tilted 20 degrees.
Why is A DNA important to know?
A DNA is seen in single-stranded RNA molecules that fold
back on themselves. A DNA is also seen in DNA-RNA
hybrids. Low humidity causes it to form from B DNA
Z-DNA
1. Left handed helix
2. 18 Angstron diameter
3. No major groove
4. 12 base pairs per turn
5. Repeating units is a dinucleotide dRY or dYR:
d(GC) d(CG) d(AC) d(GT)
6. Formation also depends on high salt to block charge
repulsion
DNA Dialogue
What forces hold a typical DNA molecule together?
ANS: Hydrogen bonds between bases either through or
or around the axis and base stacking
What is base stacking?
Stacking implies vertical interactions between bases as
they sit on top of one another
What sort of interactions?
Mainly van der Waal forces created by hydrophobic
interactions
Are the forces of interaction the same for all bases?
No. Stacking interactions between G and C give rise to
greater stacking energy than A to T
What does this do to the DNA?
Ans: The greater the GC content of DNA the greater
the stability, thermal stability in particular
What do you mean by thermal stability?
Two ways to view thermal stability. It could be the heat
energy required to separate or melt the strands
What else besides heat?
Thermal could reflect the strength of bonding of the two
DNA strands to one another though a combination
of both H-bonding and base stacking
How is thermal stability measured?
Next slide
Melting Point of DNA
Lower G + C
Higher G + C
A260
Temperature oC
50
Hyperchromicity
70
90
Tm (melting temperature)
Restriction Endonucleases
Endo = within
Exo = outer
1. Bacterial defense mechanism- the restriction
modification system
2. Work in conjunction with restriction methylases
3. Cleave DNA at a specific site
4. Some recognize palindromic sequences
5. The major tool of the cloner
Palindromes have centers of
symmetry
G AAT T C
C T TAA G
GA A T T C
C T TAA G
EcoR1
dood
it!
Fractionation of DNA Molecules
Electrophoresis
1. Separation is based on size (length) and shape
2. DNA (RNA) are polyanions move towards (+) pole
3. DNA can exist as a supercoiled or open chain
A. Open chain less compact, moves slower
B. Coiled more compact, moves rapidly
4. Hugh Size requires DNA to be cut to be visualized
5. Fluorescent reagent (ethidium bromide) used to
detect DNA (RNA) on a gel
6. Agarose and polyacrylamide are the gels of choice
cDNA Analysis by Electrophoresis
DNA Standards
Brain Intest Muscle
4.7a
4.7b
1.8
0.8a
0.8b
Size in
kilobases
(kb)
Missing Exon 10
Southern Blotting
The goal of blotting is to transfer a sample from
a 3D matrix of a gel to a 2D surface of paper for detection
1. Used to detect a specific DNA (gene) sequence
in a DNA fragment
2. Requires DNA on the gel to be transferred to
blotting paper (nitrocellulose)
3. Detecting nucleotide (probe) hybridizes to DNA
4. Probe labeled with radioactive phosphorous (32P)
for visualization by autoradiography
5. DNA on DNA called “Southern”
DNA on RNA called “Northern”
Antibody on protein called “Western”
Southern Blotting (DNA on DNA)
Blot
Develop
Probe
Detect
Ultracentrifugation in CsCl
Principle: A nucleic acid will seek its buoyant density
Single strand most dense (>1.9 g cm-1)
Pellets
Double strand less dense (~1.5 -1.6 g cm-1) Floats
Dideoxy
B
O
H
H
No base can add
DNA
Sequencing
via
Dideoxy
Method
Dideoxybases
are color coded
A=
G=
T=
C=
5’
TC A C T C
AGTGAG
A
G
T
G
Template
Selective
Termination
Polymerase Chain Reaction (PCR)
Reverse primer
binding site
DNA
5’
3’
Forward primer
binding site
R
F
3’
5’
(cycle 1)
95oC, anneal
F
R
R
F
Linear
Phase
(cycle 2)
95oC, anneal
R
F
F
R
R
Doubling
Phase
(cycle 3-25)