Lab 22 DNA Fingerprinting an Epidemic
(This is a product of Biorad. The explanation
and directions are taken from the Biorad
Instruction Manual with a few modifications)
Objectives:
 Explain the theory and
perform DNA
electrophoresis in an
agarose gel.
 Understand applications of restriction enzymes in forensic biotechnology.
 Perform micro lab skills using micro quantities, micropipetting, and metric
measurements.
 Apply and demonstrate mastery of the scientific method by analyzing a
scenario
 Analyze the results of the experiment
 Evaluate the data and determine an interpretation and potential actions
given the analysis.
Background
DNA fingerprinting is routinely used for many
applications as shown in the list below:
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solving crimes
setting falsely convicted prisoners free
identifying genetic diseases
determining birth parents (paternity)
differentiating organisms
identifying endangered species
identifying human remains
determining human ancestry
potential human migrations
identifying the presence of disease organisms
diagnosing infections or epidemic outbreaks
DNA is a powerful and accurate identification tool used
to distinguish between individuals, often referred to as
“profiling”. In microbiology DNA analysis is very useful
because it only requires small amounts of DNA to
identify an organism, it helps identify organisms that
do not grow or stain well, and it allows epidemiologists
to track specific strains or mutant forms of the same
species of bacteria.
The process (displayed at the right) involves using
enzymes to cut DNA at particular locations along the
length of DNA creating a soup of DNA chunks. A small
amount of the “soup” containing chunks of digested
DNA are then loaded into a gel using micropipettes
that deliver a small quantity into a small well. The gel is immersed in buffer and
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a small electrical current is run through the gel. The DNA moves from the well
along the gel. Larger pieces of DNA drop out near the well and the smaller
pieces of DNA continue to move toward the other end of the gel. These pieces
of DNA form a pattern consist with individual organisms. The picture at the left
has two areas where specimens were loaded into to sets of well.
Region where DNA soup is
loaded
The set of lines down
each lane are a unique
pattern for the DNA
loaded in the well at the
beginning of the lane.
Each line represents a
specific size of DNA. The
pattern of the lines is
unique for each individual
In this activity you will: 1) cut the DNA into fragments using restriction enzymes,
2) separate the DNA fragments using electrophoresis, then 3) stain the gel to
visualize DNA fragments, creating a DNA fingerprint.
Scenario:
(Refer to http://news.yahoo.com/s/nm/20060211/ts_nm/birdflu_nigeria_dc_2 )
Bird Flu, known as the H5N1 influenza virus strain, has sickened millions of birds
world-wide and been transmitted to some humans. Health workers are
concerned that the H5N1 could become a deadly pandemic. Since 2003 the
H5N1 strain has killed 88 people in Asia and the Middle East. Until now, human
cases have been contracted from close association with infected birds. Health
experts are concerned that the virus could mutate into a strain that could be
transmitted from human to human and kill millions
In February 2006 tens of thousands of birds in Nigeria became sick, and H5N1
was identified as the etiology. This outbreak was the first known occurrence of
Bird Flu in Africa. African farmers had to kill thousands of infected birds. Although
no human cases have been confirmed, logistical problems make it difficult to
verify that good human reporting is occurring. Abdulsalam Nasadi, an
epidemiologist working for the Health Ministry, acknowledged that there were a
few potential human cases, but information was scanty and difficult to track. The
following samples were sent to the special virology lab at CDC for analysis.
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Materials:
Electrophoresis System
Enzyme mix HindII (ENZ)I
DNA loading dye (LD)
Microtest tubes (color coded)
Pipet tips 2-20 μl
Micropipet 2-20 μl
Test tube holder (foam)
37C water bath
Agarose Gel
Buffer
Fast Stain
DNA Material:
Known DNA for Bird Flu H5N1
Sample 1- patient in Kaduna, Nigeria
Sample 2- patient at Sambawa Farms,
in Nigeria
Sample 3- patient at Sovet Farms,
Kano, Nigeria
Sample 4- patient at Phed Farms,
Kano, Nigeria
Sample 5- patient at Danube Farms,
Kano Nigeria
Procedures:
1. The agarose gels have been prepared by the lab technicians.
2. Place the tube containing the restriction enzyme ENZ on ice.
3. Label each colored micro test tube as follows, with a the correct letter (A,
B, C, D, E) and your table number:
A. Green tube - H5N1 known sample (control sample)
B. Blue tube - patient 1
C. Orange tube – patient 2
D. Violet tube – patient 3
E. Red tube – patient 4
F. Yellow tube – patient 5
4. Place the labeled tubes in a foam micro tube holder.
5. Use a fresh pipette tip for each DNA sample being careful to transfer the
correct specimen into the correctly labeled tube, and avoiding
contamination between species. Pipette 10 μl of DNA into each tube.
6. Pipette 10 μl of enzyme ENZ into each specimen. Observe the tube for
any changes.
7. Tightly cap each tube and mix components by flicking the tubes with your
fingers.
8. Place the tubes in the foam tube holder and incubate 45 minutes in a
water bath at 37C. After incubation, again observe the tubes for any
changes.
9. Gently tap the tubes again to mix. Then add 5 μl dye “LD” to each tube
using a separate tip.
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10. Place the agarose gel in the electrophoresis chamber and cover with
buffer. The wells need to be near the black (-) electrode.
11. Using a separate tip for each sample load the following samples and
amounts in the correct lanes.
Lane
Specimen
Amount
1
DNA Marker
10 μl
2
H5N1 Known specimen
3
4
Patient 1
Patient 2
20 μl
20 μl
5
Patient 3
20 μl
6
Patient 4
20 μl
7
Patient 5
20 μl
8
Patient 6
20 μl
20 μl
12. Turn on the power and electrophorese at 100V for 30 minutes.
13. Visualize the DNa by staining the gels.
A. Add 120 ml of 100x fast blue and gently agitate for 2 minutes.
B. Rinse with warm water about 10 seconds
C. Destain by washing in warm water 5 minutes
D. Draw a map of the results
Lane
1
2
3
4
O O O O
5
6
7
8
O O O O
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Lab #22 DNA Fingerprinting
Name______________________________
1. What is the basis of this investigation? What is the question the
epidemiologists are trying to answer?
2. What did the restriction enzymes actually do to the DNA?
3. What is the theory behind DNA fingerprinting?
4. Explain the results of the fingerprinting. What can you conclude?
5. What are the implications of the DNA fingerprinting results?
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