TEKNIK ANALISIS ASAM

NUKLEAT (DNA)
 OH
Phosphate
- NH2
HO P O Base
N
H +
H
O N
H
5’CH2
N N
Nucleotide O
4’ 1’
Sugar Nucleoside
3’ 2’

OH H
OH
5’Phosphate group
OH O
CH
3
3’Hydroxyl group

SUGAR-PHOS
NH2 H OH
HO P O
N
N HN N
O

B A S E
N N O
CH2 O
O CH2
O

H N O P HO
O H 2

O H OH
HO P O N
N
H2O
N
PHATE BACKB
O NH
O
N N NH2
CH2 O

S
O CH2
O

O
P HO
O H
NH2 O N
H O
H
HO P O
N
H2O
N HN
O N
N O N
5’Phosphate
O
CH2 H2
O CH2
ON

group
O

O HO
3’Hydroxyl group P
E

OH H
HO
 Gel Electrophoresis
 Gel electrophoresis is a widely used technique for
the analysis of nucleic acids and proteins
 Separates DNA (or RNA or Protein) fragments on
the basis of charge and size
 DNA is an acid, it looses protons in basic buffers,
thus it has a negative charge
 By placing DNA in a gel, then applying a voltage
accross the gel, the negatively charged DNA will
move toward the positive pole
 Large fragments lag behind while small fragments
move throught the gel relatively rapidly
 Agarose (a polysaccharide) or other gel matrices
are difficult for large DNA fragments to move
through
• DNA is negatively charged
• When placed in an electrical field, DNA will migrate toward the positive
pole (anode)
• An agarose gel is used to slow the movement of DNA and separate by
size
H O2
 

DNA

- +

Power • Polymerized agarose is porous,

allowing for the movement of DNA
Scanning Electron Micrograph
of Agarose Gel (1×1 µm) 
How fast will the DNA migrate?

-Gel electrophoresis separates DNA according to size of the DNA

-Small DNA move faster than large DNA

DNA

small
large
- +

Power
Within an agarose gel, linear DNA migrate inversely
proportional to the log10 of their molecular weight.
 Estimation of the size of DNA molecules
following restriction enzyme digestion, e.g.
in restriction mapping of cloned DNA.
 Analysis of PCR products, e.g. in molecular

genetic diagnosis or genetic fingerprinting

 Separation of restricted genomic DNA prior

to Southern transfer, or of RNA prior to

Northern transfer.
 The advantages are that the gel is easily
poured, does not denature the samples, and
is physically firmer than polyacrylamide.
The samples can also be recovered.
 The disadvantages are that gels can melt

during electrophoresis, the buffer can

become exhausted, and different forms of
genetic material may run in unpredictable
forms.
 The most important factor is the length of the DNA
molecule, smaller molecules travel farther. But
conformation of the DNA molecule is also a factor.
 Increasing the agarose concentration of a gel
reduces the migration speed and enables
separation of smaller DNA molecules. The higher
the voltage, the faster the DNA migrates. But
voltage is limited by the fact that it heats and
ultimately causes the gel to melt. High voltages
also decrease the resolution (above about 5 to 8
V/cm).
 Conformations of a DNA plasmid that has not been
cut with a restriction enzyme will move with
different speeds (slowest to fastest): nicked or open
circular, linearised, or supercoiled plasmid.
 The most common dye used for agarose gel
electrophoresis is ethidium bromide, usually
abbreviated as EtBr. It fluoresces under UV light
when intercalated into DNA (or RNA).
 Loading buffers are added with the DNA in
order to visualize it and sediment it in the gel
well. Negatively charged indicators keep track
of the position of the DNA. Xylene cyanol and
Bromophenol blue are typically used. They run
at about 5000 bp and 300 bp respectively, but
the precise position varies with percentage of
the gel. Other less frequently used progress
markers are Cresol Red and Orange G which run
at about 125 bp and 50 bp.
 Gel electrophoresis can be used for the
separation of DNA fragments of 50 base pairs
up to several megabases (millions of bases).
However, it is normally used in a range of 100
bp to 20 kbp. Typical run times are about an
hour.
 Small nucleic acids are better separated by
polyacrylamide gels,
 In general higher concentrations of agarose are
better for larger molecules; it will exaggerate
the distances between bands. The
disadvantage of higher concentrations is the
long run times (sometimes days). Instead these
gels should be run with a
pulsed field electrophoresis (PFE), or
field inversion electrophoresis.
 Agarose
◦ Agarose is purified from agar, a gelatinous substance isolated
from seaweed or red algae. Different purities of agarose are
commercially available as are agaroses with different melting
properties. High purity low melt agarose is often used if the DNA
is to be extracted from the gel.
 Buffer
◦ The most common being: tris acetate EDTA (TAE),
Tris/Borate/EDTA (TBE) and Sodium borate (SB).
TAE has the lowest buffering capacity but provides the best
resolution for larger DNA. This means a lower voltage and
more time, but a better product.
SB is relatively new and is ineffective in resolving fragments
larger than 5 kbp; However, with its low conductivity, a much
higher voltage could be used (up to 35 V/cm), which means a
shorter analysis time for routine electrophoresis.
 The gel with UV
An Agarose 'slab' gel illumination, the
prior to UV illumination, ethidium bromide
Only the marker dyes stained DNA glows pink
can be seen Digital photo of the gel.
Lane 1. Commercial DNA
Markers (1kbplus)
Making an Agarose Gel
 Agarose

D-galactose 3,6-anhydro
L-galactose

•Sweetened agarose gels have

been eaten in the Far East since
the 17th century.

•Agarose was first used in biology

when Robert Koch* used it as a
*Lina Hesse, technician and illustrator for  culture medium for Tuberculosis
a colleague of Koch was the first to  bacteria in 1882
suggest agar for use in culturing bacteria

Agarose is a linear polymer extracted from seaweed.

An agarose gel is prepared
by combining agarose
powder and a buffer Buffer➘
solution.

Flask for boiling➙

Agarose➚
 Electrophoresis Equipment

Power supply

Gel tank Cover

Electrical leads


Casting tray
Gel combs
 Bio-Rad’s
Electrophoresis
PowerPac™ Junior PowerPac™ Basic
Equipment

Electrophore PowerPac™ HC PowerPac™ Universal PowerPac™ 3000

sis Cells
• Power
Supplies
• Precast
Agarose Gels
Mini-Sub® Cell GT Wide Mini-Sub Cell GT
Gel casting tray & combs
 Preparing the Casting Tray

Seal the edges of the casting tray and put in the combs. Place the casting
tray on a level surface. None of the gel combs should be touching the
surface of the casting tray.
 Agarose Buffer Solution

Combine the agarose powder and buffer solution. Use a flask that is
several times larger than the volume of buffer.
 Melting the Agarose

Agarose is insoluble at room temperature (left).

The agarose solution is boiled until clear (right).

Gently swirl the solution periodically when heating to allow all the grains of agarose
to dissolve.
***Be careful when boiling - the agarose solution may become superheated and may
boil violently if it has been heated too long in a microwave oven.
 Pouring the gel

Allow the agarose solution to cool slightly (~60ºC) and then carefully
pour the melted agarose solution into the casting tray. Avoid air
bubbles.
Each of the gel combs should be submerged in the melted agarose solution.
When cooled, the agarose polymerizes, forming a flexible gel. It should
appear lighter in color when completely cooled (30-45 minutes).
Carefully remove the combs and tape.
Place the gel in the electrophoresis chamber.
 DNA

buffer     
wells
Anode
Cathode (positive)
(negative)

Add enough electrophoresis buffer to cover the gel to a depth of

at least 1 mm. Make sure each well is filled with buffer.
 Sample Preparation
Mix the samples of DNA with the 6X sample loading buffer. This allows
the samples to be seen when loading onto the gel, and increases the
density of the samples, causing them to sink into the gel wells.

6X Loading Buffer: 
• Bromophenol Blue (for color)
• Glycerol (for weight)
 Loading the Gel

Carefully place the pipette tip over a well and gently expel the sample.
The sample should sink into the well. Be careful not to puncture the
gel with the pipette tip.
 Running the Gel

Place the cover on the electrophoresis chamber, connecting the electrical

leads. Connect the electrical leads to the power supply. Be sure the leads
are attached correctly - DNA migrates toward the anode (red). When the
power is turned on, bubbles should form on the electrodes in the
electrophoresis chamber.
 Cathode
(-)

 wells
DNA  Bromophenol Blue
(-)


Gel

Anode
(+)

After the current is applied, make sure the Gel is running in the correct
direction. Bromophenol blue will run in the same direction as the DNA.
 DNA Ladder Standard
-
 12,000 bp

 5,000

DNA
migration  2,000
 1,650
Note: bromophenol
blue migrates at  1,000
approximately the  850
same rate as a 300 bp  650
DNA molecule  500
 400
bromophenol blue  300
 200
+  100

Inclusion of a DNA ladder (DNAs of know sizes) on the gel

makes it easy to determine the sizes of unknown DNAs.
 Staining the Gel
• Ethidium bromide binds to DNA and fluoresces under UV light,
allowing the visualization of DNA on a Gel.
• Ethidium bromide can be added to the gel and/or running buffer
before the gel is run or the gel can be stained after it has run.

***CAUTION! Ethidium bromide is a powerful mutagen and is

moderately toxic. Gloves should be worn at all times.
Safer alternatives to Ethidium Bromide

• Methylene Blue
• BioRAD - Bio-Safe DNA Stain
• Ward’s - QUIKView DNA Stain
• Carolina BLU Stain

…others

advantages disadvantages
Inexpensive Less sensitive
Less toxic More DNA needed on gel
No UV light required Longer staining/destaining time
No hazardous waste disposal
 Staining the Gel

• Place the gel in the staining tray containing warm diluted stain.
• Allow the gel to stain for 25-30 minutes.
• To remove excess stain, allow the gel to destain in water.
• Replace water several times for efficient destain.
Ethidium Bromide requires an ultraviolet light source to visualize
Visualizing the DNA (ethidium bromide)
DNA ladder DNA ladder
 1 2 3 4 5 6 7 8 
wells

 5,000 bp
 2,000
 1,650
 1,000
 850
 650
 500
 400
 300
 200
 100

+ - - + - + + -
 Visualizing the DNA (QuikVIEW stain)

DNA ladder

wells

 2,000 bp
 1,500
 1,000
 750
 500
 250
+ - - - - + + - - + - +
 Uncut plasmid DNA has several distinct
conformations which can be identified when the
uncut plasmid is electrophoresed in an agarose
gel.
◦ Supercoiled DNA (Form I), is the fastest conformation
of the uncut plasmid. Because of its compact shape, sc
DNA is the fastest moving comformation in the gel.
◦ Nicked Circle DNA (Form II) is also called relaxed circle.
Nicked circle (nc) is the slowest conformation of uncut
DNA.
◦ Linear DNA (Form III) is produced when a restriction
enzyme cuts a plasmid at only one site. On a gel the
linear DNA will run between the sc and nc
conformations, often closer to the sc band
 Figure 7­26
Copyright (c) by W. H. Freeman and
Company
 DNA can be recovered from agarose gel
◦ Electroelution : a slice of agarose containing DNA
bend is placed in a dialysis bag inside a
electrophoresis app.
◦ DNA bend is sliced agarose be melted in Na
iodide buffer bound to amembran/ resin DNA is
eluted
◦ Using low-melting agarose & +agarase
◦ Centrifugation-filtration
◦ Using QIAgen Gel Extraction kit
DNA FINGERPRINTING
PROCEDURE
Molecular Weight Determination
Fingerprinting Standard Curve: Semi-log
100,000
Size (bp) Distance (mm)

23,000 11.0
9,400 13.0 10,000

Size, base pairs

6,500 15.0 B

4,400 18.0
1,000

2,300 23.0
2,000 24.0
100
A
0 5 10 15 20 25 30
Distance, mm