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Structure and Analysis
of
DNA
©2000 Timothy G. Standish
Introduction
The Central Dogma
of Molecular Biology
Cell
DNA
Transcription
Translation
mRNA
Ribosome
Polypeptide
(protein)
©1998 Timothy G. Standish
Outline
1 How we know DNA is the genetic
material
2 Basic structure of DNA and RNA
3 Ways in which DNA can be
studied and what they tell us about
genomes
©2000 Timothy G. Standish
Historical Events
•
•
•
•
•
•
1869 Friedrich Miescher identified DNA, which he called
nuclein, from pus cells
1889 Richard Altman renamed nuclein nucleic acid
1928 Griffith discovered that genetic information could be
passed from one bacteria to another; known as the
transforming principle
1944 Avery showed that the transforming material was
pure DNA not protein, lipid or carbohydrate.
1952 Hershey and Chase used bacteriophage (virus) and
E. coli to show that only viral DNA entered the host
1953 Watson and Crick discovered the structure of DNA
was a double helix
©2000 Timothy G. Standish
Transformation Of Bacteria
Two Strains Of Streptococcus
Rough Strain
(Harmless)
Capsules
Smooth Strain
(Virulent)
©2000 Timothy G. Standish
Transformation Of Bacteria
The Griffith’s 1928 Experiment
OUCH!
+ Control
- Control
- Control
Experimental
©2000 Timothy G. Standish
Avery, MacLeod and McCarty
1944 Avery, MacLeod and McCarty repeated Griffith’s
1928 experiment with modifications designed to discover
the “transforming factor”
After extraction with organic solvents to eliminate lipids,
remaining extract from heat killed cells was digested with
hydrolytic enzymes specific for different classes of macro
molecules:
Enzyme
Transformation?
Protease
Yes
Saccharase
Yes
Nuclease
No
©2000 Timothy G. Standish
The Hershey-Chase
Experiement
The Hershey-Chase experiment showed definitively
that DNA is the genetic material
Hershey and Chase took advantage of the fact that T2
phage is made of only two classes of macromolecules:
Protein and DNA
H
H2N C C
CH2
CH2
S
CH3
H
O
H2N C C
OH
Methionine
CH2
SH
OH
O
OH
Cysteine
Some amino acids
contain sulfur, thus
proteins contain sulfur,
but not phosphorous.
HO P
NH2
O
O
OH
H
Nucleotides contain phosphorous,
thus DNA contains phosphorous,
but not sulfur.
©2000 Timothy G. Standish
S35
T2 grown in
containing media
incorporate S35
into their proteins
Using S35 Bacteria grown in
T2 attach to bacteria and
inject genetic material
normal nonradioactive media
When centrifuged,
phage protein coats
remain in the
supernatant while
bacteria form a pellet
The supernatant is
radioactive, but the
pellet is not.
Did protein enter the bacteria?
Blending causes phage
protein coat to fall off
Is protein the genetic material?
P32
T2 grown in
containing media
incorporate P32
into their DNA
Using P32 Bacteria grown in
T2 attach to bacteria and
inject genetic material
normal nonradioactive media
When centrifuged,
phage protein coats
remain in the
supernatant while
bacteria form a pellet
The pellet is
radioactive, but the
supernatant is not.
Did DNA enter the bacteria?
Blending causes phage
protein coat to fall off
Is DNA the genetic material?
A Nucleotide
Adenosine Mono Phosphate (AMP)
Phosphate
HO
H+
Nucleotide
OH
P
O
Base
N
H
O
5’CH2
4’
NH2
H
N
O
1’
Sugar
3’
OH
2’
H
OH
N
N
Nucleoside
Purines
NH2
Adenine
N
N
N
O
CH3
(DNA)
N
Guanine
NH
N
Thymine
O
NH2
Uracil
(RNA)
NH
N
O
N
N
Pyrimidines
NH
O
N
O
NH2
Cytosine
N
N
O
Base Pairing
Guanine And Cytosine
-
+
+
+
-
Base Pairing
Adenine And Thymine
+ -
Adenine
-
+
Thymine
Base Pairing
Adenine And Cytosine
+
-
-
Base Pairing
Guanine And Thymine
+
+
Some minor purine and
pyrimidine bases
©2000 Timothy G. Standish
5’Phosphate group
P
HO
NH2
O
N
O
CH2
OH
N
N
O
H
N
O
CH2
O
HO
P
O
O
N
O
CH2
OH
H
H2O
NH
N
O
HO
P
O
H
O
NH2
N
O
CH2
O
H
O
H
N
O
O
CH2
O
P
HO
H
O
OH
5’Phosphate
group
HO
CH2
3’Hydroxyl group
H2O
N
O
O
P
NH2
HO
P
O
H
O
HO
O
D
N
A
3’Hydroxyl group
OH
-
-
-
-
-
-
G
-
3.4 nm
1 nm
-
-
Minor
groove
C
G C
T A
A T
-
The Watson - Crick
Model Of DNA
G C
T A
C G
A T
Major
groove
A T
C G
G C
0.34 nm
T A
-
-
-
-
-
-
-
-
-
-
-
©2000 Timothy G. Standish
-
B DNA
Forms of the Double Helix
G
T A
Minor
groove
Z DNA
A T
C G
C
A DNA
3.9 nm
1 nm
Minor
groove
G C
T A
C G
A T
Major
groove
A T
G
T A
1.2 nm 2.8 nm
0.9 nm
6.8 nm
Major
groove
0.57 nm
C G
C
0.26 nm
0.34 nm
10.4 Bp/turn
+34.6o Rotation/Bp
11 Bp/turn
+34.7o Rotation/Bp
12 Bp/turn
-30.0o Rotation/Bp
©2000 Timothy G. Standish
.
©2000 Timothy G. Standish
.
A-DNA:1. Large hole in
center
2. Sugar phosphate backbone
is at the edge
3. Bases are displaced
towards edge
B-DNA-1. Bases in center (no
hole)
2. Phosphates at periphery
Z-DNA-1. Bases present
throughout the matrix of
the helix
2. No exclusive domains for
either bases or backbone
3. Left hand helix
©2000 Timothy G. Standish
Biological Significance
A-DNA-occurs only in dehydrated samples of DNA, such
as those used in crystallographic experiments, and possibly
is also assumed by DNA-RNA hybrid helices and by
regions of double-stranded RNA.
Z-DNA has been found, it is commonly believed to
provide torsional strain relief (supercoiling) while DNA
transcription occurs. The potential to form a Z-DNA
structure also correlates with regions of active transcription
©2000 Timothy G. Standish
Even More Forms Of DNA
C-DNA:
– Exists only under high dehydration conditions
– 9.3 bp/turn, 0.19 nm diameter and tilted bases
B-DNA appears to be the
– Occurs in helices lacking guanine most common form in
– 8 bp/turn
vivo. However, under
some circumstances,
E-DNA:
– Like D-DNA lack guanine
alternative forms of DNA
– 7.5 bp/turn
may play a biologically
P-DNA:
significant role.
D-DNA:
– Artificially stretched DNA with phosphate groups found inside
the long thin molecule and bases closer to the outside surface of
the helix
– 2.62 bp/turn
©2000 Timothy G. Standish
Certain DNA sequences adopt
unusual structures
Palindrome: The term is applied to regions
of DNA with inverted repeats of base
sequence having twofold symmetry over
two strands of DNA. Such sequences are
self-complementary within each strand and
therefore have the potential to form hairpin
or cruciform (cross-shaped) structures
©2000 Timothy G. Standish
Certain DNA sequences adopt
unusual structures
Mirror repeats :When the inverted repeat
occurs within each individual strand of the
DNA, the sequence is called a mirror
repeat.
Mirror repeats do not have complementary
sequences within the same strand and
cannot form hairpin or cruciform structures.
©2000 Timothy G. Standish
Certain DNA sequences adopt
unusual structures
©2000 Timothy G. Standish
.
©2000 Timothy G. Standish
.
©2000 Timothy G. Standish
keto-enol tautomerism
keto-enol tautomerism
refers to a chemical
equlibrium between a keto
form (a ketone or an
aldehyde) and an enol (An
alcohol)
In DNA, the nucleotide
bases are in keto form.
Rare enol tautomers of the
bases G and T can lead to
mutation because of their
altered base-pairing
properties.
©2000 Timothy G. Standish
Triplex DNA
Nucleotides participating in a WatsonCrick base pair can form a number of
additional hydrogen bonds, particularly
with functional groups arrayed in the
major groove. For example, a cytidine
residue (if protonated) can pair with
the guanosine residue of a G-C
nucleotide pair.
©2000 Timothy G. Standish
Triplex DNA
The N-7, O6, and N6 of purines, the atoms that
participate in the hydrogen bonding of triplex
DNA, are often referred to as Hoogsteen
positions, and the non-Watson-Crick pairing is
called Hoogsteen pairing.
The triplexes form most readily within long
sequences containing only pyrimidines or only
purines in a given strand
Four DNA strands can also pair to form a tetraplex
©2000 Timothy G. Standish
.
©2000 Timothy G. Standish
H-DNA
A particularly exotic DNA structure, known
as H-DNA, is found in polypyrimidine or
polypurine tracts that also incorporate a
mirror repeat. A simple example is a long
stretch of alternating T and C residues
©2000 Timothy G. Standish
H-DNA
©2000 Timothy G. Standish
Structure of RNA
The single strand of RNA tends to assume
a right-handed helical conformation
dominated by base stacking Interactions
,which are strongest between two purines
The purine-purine interaction is so strong that
a pyrimidine separating two purines is often
displaced from the stacking pattern so that
the purines can interact
©2000 Timothy G. Standish
Structure of RNA
RNA can base-pair with complementary
regions of either RNA or DNA. For DNA:
G pairs with C and A pairs with U ,however
base pairing between G and U is fairly
common in RNA.
Where complementary sequences are
present, the predominant double-stranded
structure is an A-form right-handed double
helix.
Hairpin loops form between nearby selfcomplementary sequences.
©2000 Timothy G. Standish
.
short base sequences (such as UUCG) are
often found at the ends of RNA hairpins and
are known to form particularly tight and
stable loops.
Additional structural contributions are made
by hydrogen bonds that are not part of
standard Watson-Crick base pairs. For
example, the 2-hydroxyl group of ribose can
hydrogen-bond with other groups.
rRNA has a characteristic secondary
structure due to many intramolecular Hbonds
©2000 Timothy G. Standish
Structure Of t-RNA
©2000 Timothy G. Standish
Denaturation and Renaturation
Heating double stranded DNA can overcome the
hydrogen bonds holding it together and cause the
strands to separate resulting in denaturation of
the DNA
When cooled relatively weak hydrogen bonds
between bases can reform and the DNA renatures
Denatured DNA
ATGAGCTGTACGATCGTG
ATGAGCTGTACGATCGTG
TACTCGACATGCTAGCAC
Double stranded DNA
ATGAGCTGTACGATCGTG
TACTCGACATGCTAGCAC
TACTCGACATGCTAGCAC
Single stranded DNA
Double stranded DNA
©2000 Timothy G. Standish
Denaturation and Renaturation
DNA with a high guanine and cytosine content has relatively more
hydrogen bonds between strands
This is because for every GC base pair 3 hydrogen bonds are made
while for AT base pairs only 2 bonds are made
Thus higher GC content is reflected in higher melting or
denaturation temperature
ACGAGCTGCACGAGC
TGCTCGACGTGCTCG
ATGATCTGTAAGATC
TACTAGACATTCTAG
67 % GC content High melting temperature
33 % GC content Low melting temperature
ATGAGCTGTCCGATC
TACTCGACAGGCTAG
50 % GC content - Intermediate melting temperature
©2000 Timothy G. Standish
Determination of GC Content
Comparison of melting temperatures can be used to
determine the GC content of an organisms genome
To do this it is necessary to be able to detect whether DNA
is melted or not
Absorbance at 260 nm of DNA in solution provides a means
of determining how much is single stranded
Single stranded DNA absorbs 260 nm ultraviolet light more
strongly than double stranded DNA does although both
absorb at this wavelength
Thus, increasing absorbance at 260 nm during heating
indicates increasing concentration of single stranded DNA
©2000 Timothy G. Standish
Determination of GC Content
1.0
Tm is the
temperature
at which half
the DNA is
melted
OD260
Single
stranded
DNA
Relatively
low GC
content
Relatively
high GC
content
Tm = 75 oC
Tm = 85 oC
Double
stranded
DNA
0
65
70
75
80
85
Temperature (oC)
90
95
©2000 Timothy G. Standish
GC Content Of Some Genomes
Organism
% GC
Homo sapiens
39.7 %
Sheep
42.4 %
Hen
42.0 %
Turtle
43.3 %
Salmon
41.2 %
Sea urchin
35.0 %
E. coli
51.7 %
Staphylococcus aureus
50.0 %
Phage l
Phage T7
55.8 %
48.0 %
©2000 Timothy G. Standish
Hybridization
The bases in DNA will only pair in very specific ways, G with C and
A with T
In short DNA sequences, imprecise base pairing will not be tolerated
Long sequences can tolerate some mispairing only if -G of the
majority of bases in a sequence exceeds the energy required to keep
mispaired bases together
Because the source of any single strand of DNA is irrelevant, merely
the sequence is important, DNA from different sources can form
double helix as long as their sequences are compatible
Thus, this phenomenon of base pairing of single stranded DNA
strands to form a double helix is called hybridization as it may be
used to make hybrid DNA composed of strands which came from
different sources
©2000 Timothy G. Standish
Hybridization
DNA from source “X”
CTGATGGTCATGAGCTGTCCGATCGATCAT
TACTCGACAGGCTAG
Hybridization
TACTCGACAGGCTAG
DNA from source “Y”
©2000 Timothy G. Standish
Hybridization
Because DNA sequences will seek out and hybridize with other
sequences with which they base pair in a specific way much
information can be gained about unknown DNA using single
stranded DNA of known sequence
Short sequences of single stranded DNA can be used as “probes” to
detect the presence of their complimentary sequence in any number
of applications including:
– Southern blots
– Northern blots (in which RNA is probed)
– In situ hybridization
– Dot blots . . .
In addition, the renaturation or hybridization of DNA in solution can
tell much about the nature of organism’s genomes
©2000 Timothy G. Standish
Reassociation Kinetics
An organism’s DNA can be heated in solution until it
melts, then cooled to allow DNA strands to reassociate
forming double stranded DNA
This is typically done after shearing the DNA to form
many fragments a few hundred bases in length. The larger
and more complex an organisms genome is, the longer it
will take for complimentary strands to bum into one
another and hybridize
Rate of reassociation is proportional to concentration of
the two homologus dissociated strands.
Reassociation follows second order kinetics:
, now integrate this equation
dt/dc = -kc2
©2000 Timothy G. Standish
Reassociation Kinetics
The following equation describes the second order
rate kinetics of DNA reassociation:
Concentration of
single stranded
DNA after time t
Initial
concentration of
single stranded
DNA
C
1
=
Co 1 + kCot
Second order
rate constant
(the important
thing is that it is
a constant)
Co (measured in
moles/liter) x t
(seconds). Generally
graphed on a log10
scale.
Cot1/2 is the point at
which half the initial
concentration of single
stranded DNA has
annealed to form
double-stranded DNA
©2000 Timothy G. Standish
Cot 0.5 value
Cot0.5 value is proportional to complexity of
the genome.
A plot of C/Co against Cot is called
Cot curve and it provides information about
complexity of a genome.
©2000 Timothy G. Standish
Genome complexity
Complexity is the minimum length of DNA
that contains a single copy of all the single
reiterated sequences that are represented
within the genome.
Complexity of a genome is equal to its
molecular mass only if a genome has
unique nucleotide sequences (repetitive
sequences absent).
©2000 Timothy G. Standish
example
# For a hypothetical DNA-1 having three
nucleotide sequences, N1, N2, N3.
Molecular mass=N1+N2+N3
Complixity=N1+N2+N3
# For a hypothetical DNA-2 having 103 copies of
N1 ,105 copies of N2 & 1 copy of N3.
Molecular mass= 103 N1+ 105 N2 + N3
Complixity=N1+N2+N3
©2000 Timothy G. Standish
Reassociation Kinetics
1.0
Fraction
remaining
singlestranded
(C/Co) 0.5
0
Higher Cot1/2
values indicate
greater
genome
complexity
Cot1/2
10-4 10-3 10-2 10-1
1
101
102 103
Cot (mole x sec./l)
104
©2000 Timothy G. Standish
Reassociation Kinetics
1.0
Prokaryotic DNA
Fraction
remaining
Repetitive
singleDNA
stranded
(C/Co) 0.5
Unique
sequence
complex
DNA
Eukaryotic DNA
0
10-4 10-3 10-2 10-1
1
101
102 103
Cot (mole x sec./l)
104
©2000 Timothy G. Standish
Repetitive DNA
Organism
% Repetitive DNA
Homo sapiens
21 %
Mouse
35 %
Calf
42 %
Drosophila
70 %
Wheat
42 %
Pea
52 %
Maize
60 %
Saccharomycetes cerevisiae
5%
E. coli
0.3 %
©2000 Timothy G. Standish
©2000 Timothy G. Standish