University of Pittsburgh at Bradford
Science In Motion
Biology Lab 002
DNA Profiling
An Exercise In Forensic Science
(Short Version using pre-digested DNA)
Safety Notes:
1. This lab requires aseptic techniques. The micropipet tips and the Eppendorf micro-tubes given to
you are sterile. Do not touch the tips and handle the micro-tubes with caution.
2. To avoid contamination, a sterile micropipet tip can only be used for one DNA sample. Eject the
tip into a beaker of Clorox and then use a new one for each new DNA sample.
3. Wear rubber gloves, preferably for the entire procedure, but definitely during staining.
Introduction and Background:
A DNA fingerprint was first introduced as evidence in an U.S. court in 1988. It is considered by
most to be the greatest advance in forensic science since the first use of the distinctive human fingerprint
in the late 1800s.
In 1984, Sir Alec Jeffreys discovered multiple copies of short sequences of nucleotides in the noncoding region of the human genome. These repeating sequences, called VNTRs (variable number of
tandem repeats), are different and at different loci in each person. DNA fingerprinting uses these
differences to identify individuals.
In this lab you will use police lab techniques to learn if the evidence collected supports the
innocence or guilt of five murder suspects. The technique uses specific restriction enzymes to cut DNA
into fragments of various lengths. Cut DNA samples (DNA digests) are then injected into the wells of an
agarose gel and run in an electrophoresis chamber to separate the fragments. Since each person’s DNA is
unique, his or her DNA will leave a fingerprint shown as bands on the gel. To save time, this lab uses
DNA that has already been cut with restriction enzymes.
PCR - polymerase chain reaction is a technique used to create millions of copies of DNA from a
small quantity such as you might find in a few cells. This is accomplished by placing the
DNA sample into a machine called a thermocycler along with enzymes, nucleotides, and
other pieces of predesigned DNA, which initiate the production of millions of copies of the
original sample in a few hours.
Restriction Enzymes - specific proteins, such as EcoRI and Pst I, which cut DNA molecules at
specific base sequences to yield DNA fragments of various lengths. These enzymes were
discovered in bacteria and act as “chemical scissors”. Recognition sequences for:
EcoRI
GAATTC
CTTAAG
Adapted From BIO-RAD: DNA Fingerprinting
Pst I
CTGCAG
GACGTC
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Agarose Gel - contains microscopic pores, which act as a molecular sieve. The sieving properties
of the gel influence the rate at which a molecule migrates. Smaller molecules move
through the pores more easily than larger ones. Hot liquid agarose is poured into an
electrophoresis tray with the gel comb in position. Impressions from the teeth of the gel
comb make the “wells,” which will be filled with DNA samples. Gels may be melted and
reused.
Gel Electrophoresis - uses electrical current to separate DNA fragments of different length on a
gel. DNA fragments (- in electrical charge) are attracted to the + pole in an electric field.
Linear duplex DNA migrates at a rate inversely proportional to the log10 of its molecular
weight. The larger the fragment the slower it migrates. Therefore, larger fragments do not
migrate very far from the well, while smaller pieces of DNA move farther.
The Crime
Cindy Kaiser, age 24, was found stabbed to death in the kitchen of her second floor apartment.
Police detectives discovered skin under one of her fingernails and reasoned that she might have scratched
her attacker before she was killed. No motive for the killing has been determined.
Five suspects were discovered to have fresh scratches:
Suspect 1 - Martin Jones - a disgruntled neighbor who was seen leaving Cindy’s
apartment that afternoon.
Suspect 2 - Harold Kaiser - was the victim’s ex-husband. Harold had a scratch on his
face. According to Harold, he cut himself while shaving. Harold said that he was sleeping
by himself at his home at the time of the murder.
Suspect 3 - Kelton Peterson - was the victim’s boyfriend. Kelton had a scratch on his
forearm, which he said happened while he was hunting grouse.
Kelton’s alibi cannot be supported.
Suspect 4 - Karen Spears - a co-worker to whom Cindy owed money to and who was
seen arguing with Cindy at work the day before. Karen had a scratch on her neck she
claims was caused by her cat.
Suspect 5 - Jack Ripper - a homeless man who lives in the ally behind Cindy’s apartment
and who was found carry a knife he claims to have found in the ally. Jack is always
suspect for crimes occurring in the neighborhood.
Forensic Procedure
The DNA from the skin found under Ms. Kaiser’s fingernail was cut with two different restriction
enzymes (EcoRI and Pst I) and amplified using the polymerase chain reaction (PCR) technique. Identical
treatment was carried out on DNA from cells taken from the five suspects. After PCR was completed, the
police lab placed the DNA samples in the following containers.
Adapted from BIO-RAD: DNA Fingerprinting
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DNA SAMPLES
Microtube Color
Label
DNA Source
Green
Blue
Orange
Violet
Red
Yellow
CS
S1
S2
S3
S4
S5
Crime scene
Suspect 1
Suspect 2
Suspect 3
Suspect 4
Suspect 5
Your lab group will use police lab electrophoresis techniques to make an agarose gel, load the
wells, and run the gels. You will then compare the DNA fingerprints to see if the evidence supports the
guilt of one or none of the suspects.
Objectives:
The learner will be able to correctly use police DNA lab techniques to collect evidence for a possible court
action. They should be able to...
1. make an agarose gel.
2. correctly operate a micropipet.
3. load, and run DNA samples on an agarose gel.
4. read and interpret the DNA fingerprint.
Materials:
Day 1 (optional)
microwave oven
Per Lab Group
1 agarose pour
1 comb
electrophoresis gel tray
casting gates
Day 2
2 power supplies
Per Lab Group
1 electrophoresis chamber
1 prepared gel
1 micropipet
micropipet tips
labeling tape
toothpicks
plastic bags or wrap
TAE buffer
loading dye
micro-tube rack with DNA tubes:
clear – DNA marker - M
green – crime scene - CS
blue – suspect 1 – S1
orange – suspect 2 – S2
violet – suspect 3 – S3
red – suspect 4 – S4
yellow – suspect 5 – S5
staining tray
Bio-Safe DNA stain
Latex gloves
Day 3
light box
Per Lab Group
staining tray
distilled water
latex gloves
collection
beaker
Adapted from BIO-RAD: DNA Fingerprinting
3
Procedure - Day 1
Making the Gel (This step may have already been done)
1. Loosen caps and heat agarose pours in microwave. Make sure that all of the agarose is melted.
2. Explanation & background from teacher.
3. Carefully seal ends of gel tray with casting gates and tape.
4. Carefully align comb and place tray out of the way on the lab table so that when the hot agarose is
poured it will be undisturbed.
5. When agarose pours are still hot (but cool enough to touch) remove cap and carefully pour hot agarose
into the tray. Make sure that your comb is aligned correctly. The agarose should cover ½ to ¾ of the
length of the teeth of the comb. Use a toothpick to move large bubbles or debris towards far end of
tray (away from comb).
6. Allow 10 - 15 minutes for gel to solidify. Do not bump or disturb tray during this time.
7. Familiarize yourself with and practice using micropipet while gels cool.
8. If time permits carefully remove comb from gel and cover tray with plastic bag for later use.
Procedure - Day 2:
Loading and Running the Gel / Staining the Gel
1. Add 5l of the loading dye “LD” to each of the colored microtubes containing digested DNA but not
to the DNA marker, which is in the clear microtube. Use a new tip for each sample.
2. Close the caps on the tubes and gently flick the tubes with your finger to mix. Pulse spin the tubes in
the centrifuge to bring the contents to the bottom of the tube.
3. Unwrap gel, cut a notch in the upper left hand corner and place it in the tray in the electrophoresis
chamber. The wells must be at the (-) cathode end of the box, where the black lead is connected.
The notch allows you to identify the wells in the gel.
4. Fill electrophoresis chamber with TAE buffer (this can be reused) until it just covers the top surface of
the gel.
5. Using the micropipet, load 10 l of the DNA marker from the clear tube into lane 1 of the gel. *Gels
are read from left to right. The first sample is loaded in the well at the left hand corner of the
gel by the notch.
Adapted from BIO-RAD: DNA Fingerprinting
4
Using a new tip for each sample, load 20 l of the digested DNA samples into the wells as follows:
Lane 2:
green, CS
Lane 3:
blue, S1
Lane 4:
orange, S2
Lane 5:
violet, S3
Lane 6:
red, S4
Lane 7:
yellow, S5
Important: A different micropipet tip should be used for each DNA sample. You will need at least
7 tips. Contaminated evidence will not hold up in court. Record the locations of samples on your
lab write-up. A good scientist keeps accurate records.
6.
7. Secure the lid on the gel box. The lid will attach to the base in only one orientation: red to red and
black to black. Connect the electrical leads to the power supply.
8. Turn on the power supply. Set it for 100 V (never set the voltage for more than 125V), and push the
RUN button. Electrophorese the samples for 30-40minutes. *Always use caution when working
with electricity to avoid electrical shock.
9. Record the time that you started. DNA fragments will be drawn toward the positive pole by the
electricity. Their progress can be followed by watching the loading dye in the DNA samples. The
electrophoresis process should be stopped when the purple dye is about 0.5 inches from the end of the
gel. Turn off the power by pressing the RUN button and remove the lid from the gel box.
10. Stop back in later in the day, or your instructor may do this for you, to begin the staining process.
a. Carefully remove the gel tray and the gel from the gel box. The gel is very slippery so use
caution not to spill it! Nudge the gel off the tray with your thumb and carefully slide it into
your staining tray.
b. Pour enough Bio-Safe DNA stain (this can be reused) into your staining tray to thoroughly
cover your gel. Carefully seal the cover onto your tray to avoid any spills. Shake (gently)
intermittently. Let sit overnight until your next class period.
Procedure - Day 3:
Destaining and Analyzing the Gels
1. Wearing gloves, carefully pour off the DNA stain into your collection container. Be careful not to
break or spill the gel during this process.
2. Destain the gel by pouring enough distilled water into the staining tray to completely cover the gel.
Allow this to sit for approximately 15 minutes with occasional gentle shaking.
3. Carefully remove the gel from the staining tray (the destaining rinse can be disposed safely down a
drain). Observe and analyze your results. Place the gel on the light box for better visualization.
4. For best results, the gel should be placed in a baggie and placed in a refrigerator overnight. A stained
gel may be stored inside a sealed plastic bag and placed in the refrigerator for several weeks. Do not
freeze. Stained gels can be safely disposed of in a wastebasket when analysis is completed.
5. Does the DNA fingerprint of any of the suspects match the DNA taken from the crime scene?
Adapted from BIO-RAD: DNA Fingerprinting
5
Name________________________
DNA Profiling
Student Evaluation
1. Describe the samples of DNA (physical properties). Is there any observable difference between the
samples of DNA?
2. The electrophoresis apparatus creates an electrical field (positive and negative ends of the gel). DNA
molecules are negatively charged. To which pole of the electrophoresis field would you expect the
DNA to migrate (+ or -)? Explain.
3. After the DNA samples are loaded in wells, they are “forced” to move through the gel matrix. Which
size fragment (large vs small) would you expect to move toward the opposite end of the gel most
quickly and therefore travel the farthest? Explain.
4. What are we trying to determine through this process of DNA electrophoresis?
5. Make a sketch of your gel showing the bands of DNA fragments that appeared during the procedure
you just completed.
6. Do any of your suspect samples appear to have EcoRI or PstI recognition sites at the same location as
the DNA from the crime scene? (a positive here is indicated by similar band patterns being created)
7. Based on the above analysis, do any of the suspect samples of DNA seem to be from the same
individual as the DNA from the crime scene? Describe the scientific evidence that supports your
conclusion.
Adapted from BIO-RAD: DNA Fingerprinting
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