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Cell Biology 2016/2017
Exercise 3
Aim of exercise
The aim of the exercise is to determine whether patients who donated biological material are
infected with high-risk human papillomavirus types. To achieve this goal DNA isolated from swabs will be
subjected to restriction with endonucleases.
Products of restriction will be separated in an agarose gel, and the band pattern of each sample will
be compared with a laboratory standard to determine the virus type.
Human papillomavirus
HPVs (human papillomaviruses), are a group of more than 150 related DNA viruses. More than 40
of these viruses can be easily spread through direct skin-to-skin contact during vaginal, anal, and oral sex.
HPV infections are the most common sexually transmitted infections in the United States. In fact, more than
half of sexually active people are infected with one or more HPV types at some point in their lives. Recent
research indicates that, at any point in time, 42.5 percent of women have genital HPV infections, whereas
less than 7 percent of adults have oral HPV infections.
Sexually transmitted HPVs fall into two categories:
• Low-risk HPVs - do not cause cancer but can cause skin warts on or around the genitals or anus
(HPV types 6 and 11 cause 90 percent of all genital warts);
• High-risk or oncogenic HPVs - can cause cancer (at least a dozen high-risk HPV types have been
identified; HPV types 16 and 18, are responsible for about 70% of HPV-caused cancers).
Virtually all cervical cancers are caused by HPV infections. HPV also causes anal cancer, with about 85%
of all cases caused by HPV-16. HPV types 16 and 18 have also been found to cause close to half of
vaginal, vulvar, and penile cancers [Hariri et al. J Infect Dis 2011; Gillison et al. JAMA 2012].
One of the methods, which can be used to discriminate between HPV viruses is RFLP (Restriction
Fragment Length Polymorphism – utilizing restriction enzymes) combined with gel electrophoresis!
Restriction digestion of DNA samples
In 1968, Dr. Werner Arber at the
University of Basel, Switzerland and Dr.
Hamilton Smith at the Johns Hopkins University,
Baltimore, discovered a group of enzymes in
bacteria, which when added to any DNA will
result in the breakage [hydrolysis] of the sugarphosphate bond between certain specific
nucleotide bases [recognition sites]. This causes
the double strand of DNA to break along the
recognition site and the DNA molecule becomes
fractured into two pieces. These molecular
scissors or “cutting” enzymes are restriction
endonucleases.
Restriction enzymes can be used to
differentiate between DNA samples as well as
detect changes in DNA sequence (mutations,
SNPs).
Agarose gel electrophoresis
Agarose gel electrophoresis separates DNA fragments by size. DNA fragments are loaded into an
agarose gel slab, which is placed into a chamber filled with a conductive buffer solution. A direct current is
passed between wire electrodes at each end of the chamber. Since DNA fragments are negatively
charged, they will be drawn toward the positive pole (anode) when placed in an electric field. The matrix of
the agarose gel acts as a molecular sieve through which smaller DNA fragments can move more easily
than larger ones. Therefore, the rate at which a DNA fragment migrates through the gel is inversely
proportional to its size in base pairs. Over a period of time, smaller DNA fragments will travel farther than
larger ones. Fragments of the same size stay together and migrate in single bands of DNA. These bands
will be seen in the gel after the DNA is stained [source: BIORAD].
I. Restriction reaction
*before you start, set the thermomixer at 37°C
Materials:
DNA, restriction enzymes, thermomixer, pipettes, eppendorf tubes, loading dye
Reagent
DNA
enzymes mixture
buffer
H2O
1.
1.
2.
3.
4.
5.
6.
7.
Stock
Final concentration/
concentration
amount
20 ng/ul
100ng
2 U/ul
0.5U/ul
10x (concentrated)
1x (concentrated)
top up to 10ul
Final volume
Using a fresh tip for each reagent combine all in a fresh eppendorf tube in the given order:
water -> buffer -> enzyme -> DNA
Seal tube with the cap and label it.
Put the tube on a vortex to ensure proper mixing of reagents.
Spin down the tube in a small bench centrifuge.
Place samples into a thermomixer (set to 37 ºC) and incubate for 30 min.
After 40 min take the samples out of the thermomixer and add 5ul of the electrophoresis loading
Dye and mix gently by pipetting up and down.
II. Electrophoresis
Materials:
agarose (powder), ethidium bromide, TBE buffer 1x, Erlenmeyer flask, analytical scale, microwave, gel
casting plate, comb, electrophoresis tank, power supply
Part 1: Gel preparation
1. Calculate the amount of agarose necessary to make 50ml of 1%
solution in TBE buffer.
2. Using the scale, weigh the necessary amount of agarose.
3. Measure 50ml of TBE buffer and pour it into an Erlenmeyer flask (A).
4. Put agarose into Erlenmeyer flask and swirl it.
5. Put the flask in a microwave and heat it for 5 min, making sure it does
not overboil.
6. Cool the solution in RT (wait app. 10 min).
7. In the mean time prepare the casting plate (B) and pour TBE buffer into
the electrophoretic tank (C).
8. Once the agarose is cool add 1ul of provided ethidium bromide to
Erlenmeyer flask and swirl it.
9. Pour the agarose into the casting plate.
10. Put an electrophoretic comb in the casting plate in order to create wells
in the gel.
11. Leave the gel to cool down (you will know it’s ready by the milky color).
A
Part 2: Electrophoresis
1. Once the gel is ready remove the comb and place the plate in the tank filled with TBE buffer.
2. Place your samples in the gel by carefully filling the wells.
3. Cover the tank with a lid and connect it to the power supply.
4. Run electrophoresis for app. 30 min applying 90V.
5. Analyze the gel using GBOX gel imaging system.
B
C