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ASAM NUKLEAT

Perbedaan struktur gula antara RNA dan DNA


2-deoxyribose sugars
Phosphodiester linkages
Directional chain (5 to 3)
4 Bases
purines: adenine & guanine
pyrimidines: cytosine & thymine

DNA is a polymer of
2-deoxyribonucleotides
pGCTA
5 end
3 end
C
G
T
A
OPO-CH
2

O
H
2
N-C

C
C

HN

N
N

CH

C

O

N

O
O
O P O CH
2

O
O
C
N
N

CH C
CH

NH
2

NH
2

C

C N

N
N

CH

C

N

HC

O
O
O P O CH
2

O
OH

O
O
O P O CH
2

O
N
C

C
O
HN

CH C
O
CH
3

1
2 3
4
5
3
RNA is a polymer of
ribonucleotides
ribose sugars
Phosphodiester linkages
Directional chain (5 to 3)
4 Bases
purines: adenine & guanine
pyrimidines: cytosine & uracil
pGCUA
C
G
U
A
5 end
3 end
1
2 3
4
5
3
OH
PO-CH
2

O
H
2
N-C

C
C

HN

N
N

CH

C

O

N

O
O
O P O CH
2

O
O
C
N
N

CH C
CH

NH
2

OH
O
O
O P O CH
2

O
N
CH

C
O
HN

CH C
O
OH
NH
2

C

C N

N
N

CH

C

N

HC

O
O
O P O CH
2

O
O-H

OH
RNA is easily hydrolyzed under alkaline conditions
The reaction proceeds through a 2,3-cyclic monophosphate intermediate.
Enzymatic hydrolysis of RNA by RNase proceeds through a similar intermediate.
Because DNA lacks the 2-OH group, it is stable under alkaline conditions.
.
.
.
.
O P O-CH
2

O
O
O
N
OH
O
O P O CH
2

O
O
N
OH
O
O P O
O
...
OH
O
N
OH
O
O P O
O
...
HOCH
2
O
.
.
.
.
O P O-CH
2

O
O
N
O O
P
O
O
H
+
.
.
.
.
O P O-CH
2

O
O
O
N
OH
O
O P OH
O
mixture of 2- and
3- monophosphate
derivatives
H
2
O
shortened
RNA
RNA
The phosphate groups
of DNA and RNA are
negatively charged
A phosphodiester group has a
pK
a
of about 1, and so will
always be ionized and negatively
charged under physiological
conditions (pH ~7).
Nucleic acids require counterions
such as Mg
2+
, polyamines,
histones or other proteins to
balance this charge.
5
3
HO-CH
2

O
N
O
O P O CH
2

O
O
N
O
O P O CH
2

O
O
N
O
O P O CH
2

O
O
O-PO
3
2
N
+
M
+
M
+
M
+
M

BENTUK DNA
B-form DNA consists of a right-handed double helix with antiparallel strands
34 (10 bp)
per turn
major groove
minor groove
major
groove
minor
3.4
per bp
These dimensions are for DNA fibers. In solution, there are ~10.5 base-pairs per turn.
5 3
5 3
Melting and Renaturation of DNA
Renaturation driven by homologous base pairing
Will also hybridize with a radiolabeled 5-ACGGCTA-3 probe.
The two strands of the double helix separate
reversibly at high temperatures
The temperature at which this
denaturation or melting
occurs depends on the pH
and salt concentration, and
increases with the GC content
of the DNA. (The curves
drawn here are schematic.)
100
80
60
40
20
0
%


D
e
n
a
t
u
r
e
d
110 100 90 80 70
Temperature /
o
C
40 50 70% GC 60
If the temperature is lowered,
the strands recombine. The
rate of reassociation is
inversely proportional to the
complexity of the DNA.
xx
Guanine
(keto form)
H
2
N-C

C
C

HN

N
N

CH

C

O

N

Thymine
(keto form)
N
C

C
O
HN

CH C
O
CH
3

N

N
C

C
OH
CH C
O
CH
3

Thymine
(enol form)
N

OH

H
2
N-C

C
C

N
N

CH

C

N

Guanine
(enol form)
The ring NH atoms
of G and T have
pK
a
values of
about 9. At
physiological pH,
about 99% of the
base is in the keto
form and 1% in the
enol form.
Tautomeric forms of G & T can cause mutations
due to mispairing during DNA replication

DNase I
DNase II

Exonuclease
Palindromic* sequences (inverted repeats) in DNA or RNA can
form hairpin or cruciform structures
inverted repeats in an antiparallel double helix
3
5
5
3
T G C G A T A C T C A T C G C A
A C G C T A T G A G T A G C G T
hairpin
C
A C
T
3 5
T
A
G
C
G
T
A
T
C
G
C
A
T
G
A
G
C
A C
T
cruciform
3 5
5 3
T
A
G
C
G
T
A
T
C
G
C
A
T
G
C
G
A
T
A
C
G
C
T
A
Mirror repeats cannot form these structures.
*A palindrome reads the same in either direction (Radar, Madam, Im Adam).
STRUKTUR
RNA
ADA PERTANYAAN ?
TERIMA KASIH
General Theory of Electrophoresis
Analytical or preparative separation of charged
molecules due to differential migration through
a matrix upon application of an electric field,
with net movement towards electrode of
opposite charge
ElectrophoresisAn Overview
Definition: The differential movement for
migration of ions by attraction or repulsion in
an electric field.
Separation of components of a mixture using an
electric field
v=Eq/f
v = velocity of molecule
E = electric field
q = net charge of molecule
f = friction coefficient
Electrophoresis- overview
cont.
Can determine the size, shape, and charge of
a molecule
Different forms of electrophoresis are used
for each of these factors independently or in
combination.
Types of Electrophoresis
Capillary
Native Polyacrylimide Gel Electrophoresis
(PAGE)
SDS-PAGE
Slab
Paper

Electrophoresis Support/Matrix
Solid matrix is most often agarose (larger
pore size) or polyacrylamide (smaller pore
size). Here, buffer provides ions for
current while maintaining constant pH

For capillary electrophoresis, buffer serves
as matrix
Components of electrophoretic
mobility
Voltage difference, E = voltage applied/distance
between electrodes; generally expressed as volts/cm
Charge on molecule, q
Frictional component, f, determined by size and
shape of molecule, pore size of matrix, viscosity of
buffer
Velocity of particle, v= Eq/f
Mobility of particle, = v/E = q/f
Approximate only- accounts for neither
interaction of particle with matrix nor
shielding of particle by buffer ions
Separation is achieved due to
differences in either or both major
components
Size/shape
Charge
Both size/shape and charge

Electrophoresis can be, but is not always,
run to an endpoint- if molecules are detected
in the matrix, an empirical endpoint chosen
such that all molecules remain in the matrix
V = IR
Voltage is a function of both current and resistance
Resistance decreases during electrophoretic run, therefore
current increases if maintaining constant voltage
To minimize this, sometimes gels are run at constant
current or at constant power (W = I
2
R)
Why minimize current increase during run?
o As current increases, power increases- much of power is
dissipated as heat
o Heat affects electrophoretic separation- diffusion increases;
samples can be sensitive to heat; buffer viscosity decreases
therefore resistance decreases and uneven heating occurs due to
greater cooling at gel edges
Outline
DNA/agarose- horizontal; separation by
size/shape
RNA/agarose- horizontal; denaturing;
separation by size
Protein/polyacrylamide- vertical
separation by size- SDS-PAGE (denaturing)
separation by size and charge-native (non-denaturing)
separation by charge (pI)- isoelectric focusing
separation by charge, then size- 2D PAGE
(denaturing)
Capillary Electrophoresis

DNA/Agarose Gel Electrophoresis
Horizontal submarine electrophoresis most
common; separation is by size
Agarose concentration 0.3-3%
Buffer most often Tris-Borate-EDTA (TBE) at 1X
or 0.5X; sometimes Tris-Acetate-EDTA (TAE) at
1X (recipes- Maniatis, Current Protocols)
Detection of DNA is generally by ethidium
bromide intercalation (dye in gel, in buffer, in
sample, or in immersion solution after run) or by
other dyes (e.g., Sypro)
Agarose
Good separation of all but smallest DNAs (<100); mid-
range to large pore size provides sieving effect; relatively
inert
Linear polysaccharide purified from seaweed extract is
composed of repeating units of agarobiose
When dissolved by heat in aqueous solution, then cooled, agarose
solution gels due to formation of inter- and intra-chain H bonds
=> The higher the concentration, the smaller the pore size
Preparing and running an agarose gel
Suspend agarose in running buffer (NOT H
2
O) to desired
concentration
Heat to boiling; once dissolved, cool to ~65
o
C; add EtBr if
desired to 1 g/ml; pour into gel tray with comb to form wells; let
set completely
Prepare DNA samples- add loading dye to 1X (provides high
density to allow sample to sink, and provides dye for monitoring
migration)
Remove comb from gel, set up in tank and submerge in buffer
Load samples by pipetting slowly through buffer into wells
Attach leads to tank and power supply; set V so I < 60 mA
DAYA PISAH
ELEKTROFORESIS GEL
Agarosa % Fragmen kb
0,5 1-30
0,7 0,8-12
1,0 0,5-10
1,2 0,4-7
1,5 0,2-3
3 0,01-0,5
Akrilamid% Fragmen kb
3,5 100
Running an agarose gel- agarose
concentration
Agarose concentration needed is determined by
sizes of DNA fragments to be separated
o for fragments between 0.3 and 3 kb, 0.7-1% are
good standard concentrations
o to separate small fragments (0.2-0.7 kb) from
one another, use 1.5-3% (smaller pore size)
o for separation of larger fragments (>3 kb and
less than 30 kb) use 0.3-0.5%
Running an agarose gel- agarose
concentration
At low agarose concentrations, very little
frictional resistance for small fragments => mobility
is similar between different sizes; for high agarose
concentrations, very little separation of large DNA
fragments of different size because high frictional
resistance for all
Separation of fragments above 20 kb is not ideal
using normal electrophoresis setup
Estimating molecular size by determining
electrophoretic mobility
Dyes provide both the means of determining endpoint of
run and the basis of determining mobility (M
r
)

and
estimated size (DNA in kb)

Bromophenol blue (BPB) and xylene cyanol are the most
common agarose gel tracking dyes. Both dyes run with
characteristic mobility that varies with gel percentage; in
general, BPB runs at 0.3-0.6 kb and xylene cyanol at 3-6
kb
Estimating molecular size by
determining electrophoretic mobility
Mobility, M
r
= distance migrated by band of
interest/distance migrated by dye front

M
r
is related to logMW or log of molecular size in bp
in a linear fashion, therefore if plot standards on
graph, one can determine masses of unknowns
Estimating molecular size by determining
electrophoretic mobility
Distance from origin in cm, or M
r

Log
MW
unknown
HIBRIDISASI
SOUTHERN
NORTHERN
WESTERN
HIBRIDISASI ASAM NUKLEAT
Tm pelacak
5-AGACTCAGAGAGAAACCC-3
4G 5C 7A 1T
Tm = (4x[4+5]) + (2x[7+1]) = 36 + 16
= 52