Download Structure and Analysis of DNA - Circle

John 5:37
37 And the Father himself,
which hath sent me, hath
borne witness of me. Ye
have neither heard his
voice at any time, nor seen
his shape.
©2000 Timothy G. Standish
Structure and Analysis
of
DNA and RNA
Timothy G. Standish, Ph. D.
©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
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.
Blending causes phage
protein coat to fall off
Did protein enter the bacteria?
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.
Blending causes phage
protein coat to fall off
Did DNA enter the bacteria?
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
+
+
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
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
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
Reassociation follows second order kinetics
©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
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