Components of DNA
Hydrogen bonding
Molecular Biology of Gene Watson et al ©
Preferred Tautomer
enol keto
Molecular Biology of Gene Watson et al © amino imino
Structure of DNA
On
in
The space-filling model
of the double helix
Molecular Biology of Gene Watson et al ©
Non-Watson-Crick Base Pairing
A T T
A T
A
Hoogsteen base pairing
EE
E loop is observedin the
Rn
Sarin ricin loop 57249 23s
which involvedin Ev Tu EF a binding
k turns pseudoknots
Bates
N
Major and Minor Grove in DNA
N
ADAM =A:T AADH = G:C
MADA =T:A HDAA= C:G
A=H-bond Acceptor
D=H bond Donor
M=Methyl group
Molecular Biology of Gene Watson et al © H=non polar hydrogen
Multiple Conformations of DNA Double Helix
Propeller twist
NaCl >2M;
MgCl2 >0.7 M
Molecular Biology of Gene Watson et al ©
Comparison of different forms of DNA
A DNA B DNA
RIVA
Molecular Biology of Gene Watson et al ©
r 5th
The axis is the center the base
1 I
it of ÉÉt
HEAD Hyped D
o o
1
OF Mon 20
CBased
plot
Kyrie K 517
o Kyrie
oh
0
I
On
0
9
o on
0 40 0
o
o
0 0
15 HIT'd
o n
4
ED
o
no
0 0 1013 OF o
the i
II
Identify Right handed and Left handed DNA
1 2 3
Properties of DNA
UV light absorbance properties : Hypochromic and hyperchromic effects
A260 is proportional to concentration, with value of 0.02 units per microL
A260 Double-stranded DNA= 1.00
E extinctincettient
song mi A260 Single-stranded DNA = 1.37
A260 Free base = 1.60
SsDNA 0 027
DNA melting curve?
Hydrogen bonding and base stacking stabilize the DNA helix structure and
work in cooperative manner
Substances that destabilize the DNA helix
FINE Urea, Formamide, alcohols,
Evidences for H-bonds and Base stacking
Cooperatively of Base stacking
helix-destabilizing melting proteins (Gene 32:T4)
SSB-Single strand binding proteins (DNA replication)
Molecular Biology David Freifelder ©
DNA melting cure
Japon
[Link] 1
Nature ofsolvent
ph of the solan
IT
ÉEÉÉÉÉ
Denaturation of DNA first order kinetics
dsDNA Second order kinetic
Renaturation of
AT E A LAY
compliment
Iole ate my stand
one tholeades
Effect of Ionic strength on DNA melting
Na+ form "clouds" of charge around the negatively charged phosphates
and effectively shield the phosphates from one another.
Molecular Biology David Freifelder ©
DNA Renaturation
q Salt concentration must be high to overcome electrostatic repulsion
between the phosphates (0.15 to 0.50 M) NaCl.
qThe optimal temperature for renaturation is 20-25° below the value of Tm
1A 1B 2 1C 1A 1B 2 1C
ATGA…..ATGA…..CCCC…..ATGA….. ATGA…..ATGA…..CCCC…..ATGA
TACT…...TACT…..GGGG….TACT….. TACT…...TACT…..GGGG….TACT…..
1A’ 1B’ 2’ 1C’ 1A’ 1B’ 2’ 1C’
Random Collision
At elevated Temp there is sampling of complementary base pairing/stacking in neighbor
Renaturation follows law of mass action (ie: concentration dependent)
DNA Hybridization
Application in Southern & Northern Blot
Efficacy of renaturation depends
upon DNA concentration
M wt=2.5 x 107
T7 Molar= 4.4x T4
M wt=1.1x 108
Molecular Biology David Freifelder © Molecular Biology of Gene Watson et al ©
in venaturation
Hybridization parameters
kinetics
1 Concentration of cations
209 below Tm
9 Incubation temperature
3 DNA concentration complexity of DNA
4 size of the fragments
nite
experimentatontwithisatfin ah
Ei mangy
Reanneling
long DNA
For molecules
must I 1 108
less
Ty 00
2 5 107 more molecules
M wot
Tf 08 renaturefaster
Chan th DNA
because con
of more
EEE
É
MEET
F
anti I
in venaturation
tybridization parameters
kinetics
1 Concentration of cations
20 C below Tm
9 Incubation temperature
3 DNA concentration
4 the complexity of DNA
size of fragments
Southern Hybridization o
r
fawn
itis
si
É a
[Link] FÉTÉ'd
ffgf
1I H 4th F
tf aug
1 it
4 a
A 4
ATAT AT AAT AT 1
[Link] EEtEIEEgEE
In
Hydroxyapatite dissolved in
ioosn phosphate
[Link] 0.129
at V0 50M
Sodium
buffer
SPB
1F
A 60
Elute at 0 519
0 0319
ds DNA
SSDNA part p
Cot eAna lysis Rate at which DNA re natures
is indicative of the length of its unique sequences
Shear the DNA into 300 10,000 top bysonication
Denature and allow it to re nature
chromatography
pass through Hydroxyapatite
elute from low salt to
high salt can
with constant measuring of A 260
This give indication of DNA is ss Is
f
Formation of dsDNA is depend upon
perfect collision of complementarysequence
collision with non complementary sequence
does not lead to renaturation on dsDNA
formation
Rate renaturation is expressed as
when Aand B
KLA CB
dff
represent complementary
singlestrand sequence
k Second order rate constant
Since A B for duplex DNA integration
above equal gives to
of
tht
TA FAI yalhere A is initial Con of A
B It is convenient to measure fraction f of
strand
unpaired
A
i f
Tag
Solving
Artifact
strand doesnot
non compliment
since the collision of
lead to rematuration A
Cg
is initial concentration of barepair
hathere Co
in solution and
base in each unique
a number of pair
Also known as DNA complexity
sequence
12
f Iffy
t
when 1 0.5
20 rate constant
Cot 112 2 12
complexity
tax
19 I
uo
u v uu r
TAVEVATAVAUTZ
this
Numberof times
fragment and sequence
observed in the genome GATACTAT CC GTA 13
Cot
Cary
Rate of renauration
CAILLATB
s s
KAJ CBI AI
flay Kt
I
cone
initial
FAI of LA
fE
f Fat
n
Where co is initial concof basepair
in south and in each
N Number of base Dna complexity
se e
unique
C0t curve
1
f=
1+C0tk/x
f=fraction of unpaired strand
x= number of unique sequence
C0= initial con of base-pairs in solution
t=time
k=second order rate constant
When f=0.5
C0t1/2 =x/k
For a given set of conditions C0t½ depends upon the complexity of the DNA (x)
When k is rate constant (single strand collision)
C-Value Paradox
human genome
is 650x
larger
E coli
than yet
encodes only N 5 h x
as
many structural
genes
DNA Topology
(cccDNA) covalently closed circular DNA: The two ends are covalently linked to form a
circular DNA molecule (topologically constrained)
Linking number (Lk)
Interwound The number of times one strand have to
be passed through the other strand in
order to completely separate the two
strands. (it is always an Integer)
Lk=Tw +Wr
(Twist) (Writhe)
cccDNA free of supercoiling
(relaxed form) is denoted as LkO
Linking differences
Measures the extent of supercoiling
Lk – LkO=DLk
Negative supercoiled If Lk <LkO and DLk <0
Positive supercoiled If Lk >LkO and DLk >0
32-36= -4
Superhelical density of DNA
Molecular Biology of Gene Watson et al © s=DLk/LkO ~0.06
Significances of DNA super-coiling
q The DNA super coil stores free energy that require strand separation, such as
DNA replication and transcription.
q Generally negative super-coiling is observed in Pro and Eukaryotic cells.
However, positive super-coiling was also found in thermophilic microorganism,
which keep their DNA from denaturation at elevated temp.
q Wrapping of DNA around nucleosome also induce negative supercoiling.
q Prokaryotes have a special type I topoisomerase known as DNA gyrase that
introduces negative supercoils and facilitate DNA supercoiling.
Ethidium increases Wr
Molecular Biology of Gene Watson et al ©
Topoisomerase
Type I Type II
Type I topoisomerases change the
linking number of DNA in steps of one.
Topoisomerases can both catenate and
decatenate circular DNA molecules.
Molecular Biology of Gene Watson et al ©
Topoisomerase-I in action
Molecular Biology of Gene Watson et al ©