Mohd Hairi Bin A.Hamid – Bsc.

Medical Laboratory Technology (Hons)

UniversitI Teknologi Mara, Malaysia – (July 2009)

Question

1) Describe a detailed used of Agarose gel @ Acrylamide gel electrophoresis in clinical

laboratory.
Agarose gel electrophoresis
Introduction/ Properties

Agarose gel electrophoresis is the easiest and commonest way of separating and analyzing
the nucleic acid especially DNA.

Agarose is a linear polysaccharide with their average molecular weight about 12000. It is the
natural colloid extracted from seaweeds and made up of the basic repeat unit agarobiose, which
consist alternating units of galactose and 3,6-anhydrogalactose. Agarose is usually used at
concentrations between 1% and 3%. Agarose gels also have very large “pore” size and are very large
molecules with the molecular weight greater than 200 kdal.

Furthermore, most agarose gels are made with between 0.7% (good resolution for large 5-
10Kb DNA fragments) and 2% good resolution for small 0.2–1kb fragments) agarose dissolved in
electrophoresis buffer. Up to 3% can be used for separating very tiny fragments but polyacrylamide
gel is more appropriate used. The agar melts at 85 °C (358 K, 185 °F) and solidifies from 32-40 °C.
(305 - 313 K, 90-104 °F).

Gel electrophoresis apparatus

An agarose gel is placed in this

buffer-filled box and electrical
current is applied via the power
supply to the rear. The negative
terminal is at the far end (black
wire), so DNA migrates toward the
camera.

Principles

It is used to look, quantify or to isolate or separate DNA or RNA fragments according to their
size and electrical charge. Typically, a DNA/ RNA molecules are digested with restriction enzymes,
and the agarose gel electrophoresis is used as a diagnostic tool to visualize the fragments. This is
achieved by moving negatively charged nucleic acid molecules through an agarose matrix with an
electric field (electrophoresis), which is shorter molecules, move faster and migrate farther than
longer ones.

The DNA or RNA bands is visualised in the gel by addition of ethidium bromide. This binds
strongly to DNA by intercalating between the bases and is fluorescent meaning that it absorbs
invisible UV light and transmits the energy as visible orange light.
 Mohd Hairi Bin A.Hamid – Bsc. Medical Laboratory Technology (Hons)
UniversitI Teknologi Mara, Malaysia – (July 2009)

ADVANTAGES OF AGAROSE GEL DISADVANTAGES OF AGAROSE GEL

 Easily poured
 Does not denature the samples
 The samples can also be recovered
 Can be processed faster than
polyacrylamide gel
 Gels can melt during electrophoresis
 Fragile, which is easily destroyed by
handling
 The buffer can become exhausted
 Different forms of genetic material may
run in unpredictable forms
 The bands formed in the agarose gel are
fuzzy and spread far apart (result from
pore size and it cannot be controlled)

Procedure used with Agarose Gel

After the gel has been prepared, use a micropipette to inject about 2.5 µl of stained DNA (a
DNA ladder is also highly recommended). Close the lid of the electrophoresis chamber and apply
current (typically 100 V for 30 minutes with 15 ml of gel). The colored dye in the DNA ladder and
DNA samples acts as a "front wave" that runs faster than the DNA itself. When the "front wave"
approaches the end of the gel, the current is stopped. The DNA is stained with ethidium bromide,
and is then visible under ultraviolet light.

1. The Agarose Gel with three slots/wells (S).

2. Injection of DNA ladder (molecular weight markers) into the first slot.
3. DNA ladder injected. Injection of samples into the second and third slot.
4. A current is applied. The DNA moves toward the positive anode due to the negative charges
on its phosphate backbone.
5. Small DNA strands move fast, large DNA strands move slowly through the gel. The DNA is
not normally visible during this process, so the marker dye is added to the DNA to avoid the
DNA being run entirely off the gel. The marker dye has a low molecular weight, and migrates
faster than the DNA, so as long as the marker has not run past the end of the gel, the DNA
will still be in the gel.
6. Add the colour marker dye to the DNA ladder
 Mohd Hairi Bin A.Hamid – Bsc. Medical Laboratory Technology (Hons)
UniversitI Teknologi Mara, Malaysia – (July 2009)

Migration of DNA Fragments in Agarose

An ethidium-stained gel with

uncut plasmid in the left lane
and the same plasmid
A pattern of
linearized at a single site in
DNA-bands
the right lane.
under UV
light
Migration of a set of DNA fragments
in three concentrations of agarose
 Mohd Hairi Bin A.Hamid – Bsc. Medical Laboratory Technology (Hons)
UniversitI Teknologi Mara, Malaysia – (July 2009)

Applications of the Agarose Gel

 Analyze DNA fragments that result from an enzyme digestion of a larger piece of DNA to
visualize and determine the size of the fragments, e.g. in restriction mapping of cloned
DNA.
 Analysis of PCR (Polymerase Chain Reactions) products, e.g. in molecular genetics diagnosis
or genetic fingerprinting
 Separation of restricted genomic DNA prior to Southern transfer or of RNA prior to
Northern transfer.
 Human Genome Project; there are four complimentary approaches are being used:
→ Genetic mapping of human genome
→ Physical mapping of human genome
→ sequencing the human genome
→ analyze the genome of other species.

(1) DNA Profiling @ DNA Fingerprinting

There are several processes involved in this technique;

a) Extracted DNA → DNA is extracted from sample (blood, semen,etc)

→ After DNA is amplified by PCR, DNA is cut into fragments of different length
Using restriction enzymes

b) Gel Electrophoresis
→ used to separate the DNA fragment according to their size
→ they separate due to both size & electrical charge
→ the smaller fragments move faster down the gel than the longer one.

c) Southern Blotting
→ the gel is treated with alkali in the buffer solution to split double-stranded DNA into single
Strand
→ A copy of DNA strand is transferred from agarose gel to nitrocellulose @ Nylon filter

d) Gene probe hybridisation

→ Blotted nitrocellulose is incubated with selected, radioactively labelled
→ DNA probes – to bind to particular bonds of DNA
→ the unbound probes are washed away

e) Autoradiography
→ the nitrocellulose paper is placed on an x-ray film & exposed to radioactivity (autoradiography)
→ the positions of the bond (fragments) to which the probe has been hybridized is shown
 Mohd Hairi Bin A.Hamid – Bsc. Medical Laboratory Technology (Hons)
UniversitI Teknologi Mara, Malaysia – (July 2009)

References

1. D R Schaberg, L S Tompkins, and S Falkow (1981), Use of agarose gel electrophoresis of

plasmid deoxyribonucleic acid to fingerprint gram-negative bacilli; retrieved from
http://www.ncbi.nlm.nih.gov/pubmed
2. S Freidenrich, R Hand (1981), he use of agarose gel electrophoresis to measure the size of
DNA molecules in crude cell lysates; retrieved from http://www.sciencedirect.com/science
3. http://www.molecularstation.com/agarose-gel-electrophoresis/
4. http://www.methodbook.net/dna/agarogel.html
5. Practical Applications of DNA Fingerprinting (200, from
http://protist.biology.washington.edu/fingerprint/dnaintro.html
6. http://askabiologist.asu.edu/expstuff/mamajis/agarosegelelectroporesis.html
7. http://en.wikipedia.org/wiki/Gel_electrophoresis
8. http://www.vivo.colostate.edu/hbooks/genetics/biotech/gels/principles.html
9. http://www.vivo.colostate.edu/hbooks/genetics/biotech/gels/agarrna.html