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Forms of DNA
B - DNA: Watson-Crick Structure
10 residues/turn
A - DNA: wider helix - tilted bases
no minor groove
RNA-DNA hybrids
Z - DNA: High salt form - left handed
antibodies to Z - DNA exist
in nature
3
Procaryotic DNA
no structural proteins bound
circular DNA
supercoiling: conserves space
easier to unwind helix
L=T+W
L = 25 + 0 L = 23 + 0 4
DNA Gyrase
Helicase
Topoisomerase
I
L = 25 - 2
5
Eucaryotic DNA
50:50 DNA:Protein
Histones: basic proteins
octamer core = (H2A, H2B, H3, H4)2
H2A H2B
=
H3 H4
P
H1
Eucaryotic Chromosome
23
Genetic Information
DNA Replication
Transcription
DNA
RNA
Gene
Translation Expression
Protei
n
24
Substrates Enzyme
Replication
DNA Polymerase
dATP
dGTP DNA
dTTP
dCTP DNA
Product
Template
25
REPLICATION
Connects dNTP’s
(3’-5’ Phosphodiester bond)
Template instructions:
parent cell’s DNA
Single copy only:
prior to cell division
DNA Gyrase +
Helicase + replication fork
SSB Protein
5
3 DNA Polymerase
The new strand is formed 5 3
29
In Real System DNA Pol
“dimers” act in tandem
5’ 3’
5’ 3’
Thymine has an equilibrium between 31
keto & enol forms
O
1
H CH3
HO N
CH3
N O N
keto
O N
enol
10,000 or
10 4
Imino form of Adenine pairs with C
NH
H N
N
H2N
N N N
N
Adenine (imino)
N N
32 Adenine (amino)
33
Fidelity of T C
Replication G
A G T A
Chance of error
C C
1 in 104 C G
T AT
C A
G G
G C
A C
C T
U G
A A
T
34
DNA Polymerase “Activities”
5’-3’ Polymerase: (normal)
3’-5’ exonuclease: removes last base
in growing strand if incorrect
T
discontinuous (lagging) strand
requires new primer
Continuation
of Replication
continuous strand
38 no additional primer
39
Okasaki Fragments
continuous
40
E coli DNA Polymerases
DNA Pol I: primer removal & gap filling
DNA Pol II: DNA repair?
DNA Pol III: main polymerase
()2 complex
proof reading processivity
5’-3’ polymerase dimerization
41
E coli DNA Polymerases
DNA Pol I: primer removal & gap filling
S-P-S-P S-P-S-P
T A
T G T G C T A C A
S-P-S-P-S-P-S-P-S-P-S-P-S-P
3’ 5’
S-P-S-P-S-P-S-P S-P-S-P-S-P
T A G C A C A
T G T G C T A
S-P-S-P-S-P-S-P-S-P-S-P-S-P
O N
O N
C
The lack of CH3 on U in DNA marks as error
and instigates removing and repair to restore C
Why both T & U?
10
Restriction Endonucleases/Methylases
Recognize and bind to specific DNA sequence
Function in bacteria is to destroy foreign DNA
1) if methylated – ignore
2) if ½ methylated – methylate other strand
3) if unmethylated - cut
4
4
5 5
4 4
2 2
2’
1 1 3 3
3 3
2
1 1
5 5
13
DNA Sequencing
Cut DNA into managable pieces
Create DNA Library of pieces
Select desired piece (Southern Blot)
Clone & amplify desired piece - PCR
Sequence cloned fragment
Southern Blot add 32P 2′ to 14
nitrocellulose blot
4
5
4
2
1 3
3
2
1
5
15
Polymerase Chain Reaction (PCR)
Heat +
0 16
5’ end O O O
|| || || A
O - P - O - P - O - P -O
|
-
O
| | |
O_ O_ O_
O
|
C
_
O - P -O O |
|
O_
19 OH
3’ end
20
3’- XXXXACGTAGCTTACC - 5’
5’-32PXXXX
Add ddTTP + dTTP, dATP, dCTP, dGTP
Get : XXXXT’
XXXXTGCAT’
XXXXTGCATCGAAT’
piece length = Primer + 1,5,10
1 2 3 4 5 6 7 8 9 10 11 12
21
3’- XXXXACGTAGCTTACC - 5’
5’ PXXXX
-32
Fragment Sizes :
ddT : 1, 5, 10
ddA : 4, 8, 9
ddC : 3, 6
ddG : 2, 7, 11, 12
22
T A C G
12
11
10
9
8
7
6
5
4
3
2
1
5’-XXXXTGCATCGAATGG
Hasil Agarose Elektroforesis