DNA Fingerprinting Laboratory
Introduction
Deoxyribonucleic acid (DNA) is present in every nucleated cell. In mammals, much of the
DNA does not code for proteins and, as a result, is not subject to the same evolutionary pressure
that makes much of the coding DNA similar, if not the same, between different individual
organisms within a species. Noncoding DNA may be found in introns or between genes, and is
often composed of repeated sequences of blocks of 2 to 5 nucleotides known as short tandem
repeats (STR). As a result of duplication mutations, the number of STR varies between individuals.
These DNA polymorphisms (as they are called by scientists and hip students) may be used to
differentiate between individuals.
In order to analyze DNA polymorphisms, it is necessary to increase the amount of the
relevant DNA sequences in the sample. This is accomplished by the use of a technique called
polymerase chain reaction, (PCR) which is somewhat like extracellular DNA replication. In the
first step of PCR, heat (rather than helicase enzyme) is used to open up the DNA strand. Because of
this, a super-special polymerase, originally isolated from the geyser-living bacterium
Thermophilus aquaticus, is used to make the complementary DNA. The sample is then cooled,
which allows primers (single-stranded pieces of DNA that guide the polymerase to the region of
interest) to bind to complementary regions of the DNA. The sample is then heated again, which
allows the polymerase to assemble nucleotide monomers to make a single strand of
complementary DNA between the primers. The cycle is then repeated, generally 20 and 40 times.
Because each newly made strand of DNA serve as a template strand for the next cycle, the PCR
process causes an exponential increase in the amount of target DNA.
Agarose gel electrophoresis is then used to visualize the PCR products. To do this, the PCR
products are mixed with a dye and placed in an agarose gel. Agarose is similar to the agar that we
have used in previous labs. An agarose gel contains many tunnels through which the DNA, driven
by an electrical current, moves. This allows us to separate the strands of DNA by size; small
fragments travel faster, larger ones slower. You might think of an agarose gel as a kind of sieve.
The techniques of PCR and gel electrophoresis are widely used in molecular biology, and
also in the field of forensics. If appropriate primers are used, it is possible to create a DNA profile
(commonly known as a DNA fingerprint) that is unique for each individual human. In this
experiment we will compare DNA profiles from three different suspects to one taken from a crime
scene in order to determine which suspect, if any, is most likely to have been the criminal. Please
bear in mind that the material we will be using is not really human DNA from a crime scene; it is
most likely PCR products made from bacterial DNA.
Procedures
1. The casting of the gel
a. Place the rubber “bumpers” on the ends of the casting tray.
b. Pour the warm gel into the casting tray and allow to cool on the windowsill for about 20
minutes until it hardens.
2. The practicing of the technique
a. Set the pipette to 30 ul and fit it with a yellow tip.
b. Push the plunger down until you first feel resistance.
c. Draw practice solution (water with blue dye) into the tip by releasing the plunger in a
controlled fashion-don’t allow it to spring up on its own.
d. Place the tip down into (but not to the bottom of) the well and slowly push down on the
plunger. After the initial resistance you will be able to push the plunger down a little
farther-this will discharge the small amount of liquid that remains in the tip.
e. Repeat a few times until you can do it smoothly and accurately.
3. The preparation of the electrophoresis rig
See the flow chart and text instructions on the following pages for an overview
a. Carefully remove the rubber bumpers from the gel casting unit-it is helpful to run a
pipette tip along them to prevent tearing of the gel.
b. Place casting tray into the electrophoresis unit
c. Add running buffer (a salt solution) into the unit until it covers the gel completely
d. Remove the plastic “comb,” being careful to avoid damaging the wells
4. The application of the samples
a. Carefully set the pipetter to 25 ul.
b. Add the samples to the wells according to the map you made in your laboratory
notebook.
c. Use a new, clean tip for each sample.
5. The turning on of the electricity
a. Make sure that the gel is placed in the electrophoresis unit so that the negatively charged
DNA will migrate toward the positive electrical terminal.
b. Check again-getting this wrong may spell disaster for your results
c. Hook up the wires from the electrophoresis rig to the power supply and turn on the
electricity. Electrons will flow.
d. Monitor the progress of your DNA periodically as you work on the rest of the lab. You
should see the blue band, which is tracking dye, moving down the gel.
6. The staining and destaining of the gel and visualization of the DNA
a. Carefully remove the gel from the casting tray and place it in a staining chamber.
b. Submerge the gel in staining solution. Wait 5 minutes
c. Dump out the used staining solution
d. Submerge the gel in distilled H20 and swoosh it around a bit.
e. Repeat (d) once or twice until the dye has been washed out of the gel.
f. Place the gel on a light box. You should see DNA bands in each lane.
g. Draw a picture of what you see. Be sure to label each lane.
7. The cleaning up of the mess
a. Rinse out casting trays. Put them back how you found them at the beginning of the lab.
b. Used pipette tips go in the trash.
c. Dump used running buffer in the appropriate container on the lab bench.
d. Dump used staining solution in the appropriate container on the lab bench.
e. Dump used destaining water down the drain.
f. Dispose of gels in trash.
g. Wipe down lab bench, organize all equipment.
Questions:
1. Which suspect, if any, most likely committed the crime?
2. Explain the evidence.
3. Under what condition might this evidence point you to the wrong person?
4. What is polymorphic DNA?
5. How does DNA of individuals within a species become polymorphic?
6. Why is it possible to use polymorphic DNA to identify individuals?
7. Why is it necessary to use PCR to amplify the target DNA before performing
electrophoresis?
8. What are some applications, beside forensic science, for DNA profiles?