DNA FINGERPRINTING LAB
Who stole the cookies from the cookie jar? A simulation
Many of the revolutionary changes that have occurred in biology over the past twenty years can be
attributed directly to the ability of scientists to manipulate DNA in defined ways. The principal tools
for this recombinant DNA technology are enzymes that can cut, mend, wind, unwind,
transcribe, repress, and replicate DNA. Restriction enzymes are the "chemical scissors" of the
molecular biologist; these enzymes cut DNA at specific nucleotide sequences. If a sample of a person's
DNA is incubated with a restriction enzyme, it is reduced to millions of DNA fragments of varying
sizes. A DNA sample from another person would have a different nucleotide sequence and would thus
be enzymatically "chopped up" into a very different collection of fragments. The DNA fragments are
separated according to size by electricity—agarose gel electrophoresis—and tagged or stained in
some fashion so that they can be visualized and studied. The resulting pattern of restriction fragments
resembles the pricing bar code used on supermarket products—small bars lined up in a column, the
largest fragments closest to the beginning of the gel, the smallest at the end of the gel.
Methods of DNA identification have been applied to many branches of science and technology, including
medicine (prenatal testing, genetic screening), conservation biology (guiding captive breeding
programs for endangered species), and forensic science. In the latter discipline, analysis of the
pattern of DNA fragments in a restriction digest, loosely called DNA typing or DNA
fingerprinting, enables us to discriminate between suspects accused of a crime or parents in a
The variation in DNA from one person to the next is so great that the probability of two people (other
than identical twins) sharing the same DNA fingerprint is essentially zero. And unlike conventional
fingerprints, which are often difficult to gather at a crime scene, a DNA fingerprint can be made from
a very small sample of blood, skin, semen - or even a single hair!
In true DNA fingerprinting, radioactive pieces of DNA called probes are added to the separated DNA
fragments. The probes are designed to bind to specific sequences on the DNA, thus marking some of the
fragments. An X-ray film placed near the gel will become exposed by the radioactivity, and black bands
will become visible on the film.
The usual: title, date, purpose, procedure & safety. You MUST also set up a table showing how much of
each reagent or solution goes into each reaction tube. It’s a good idea to include a diagram of how the
micropipet dial will look for each solution, too. Check off each solution as you put it into reaction tube.
Mr. Hardie returned to his office to find no cookies in his cookie jar. Cheek cells were taken from each
of the other Biology teachers and their DNA isolated. Skin cells were also taken from the lid of the
cookie jar. We will compare the DNA fingerprints of the teacher samples to those from the jar to find
Mr.Spence #1 Ms. Dr. Weaver #3 Cells from Jar UR
4µL enzyme 4µL enzyme 4µL enzyme 4µL enzyme 4µL dH2O
6µL buffer 6µL buffer 6µL buffer 6µL buffer 6µL buffer
2µL #1 DNA 2µL #2 DNA 2µL #3 DNA 2µL CS DNA 2µL UR DNA
D A Y 1. Restrict DNA samples and cast gels.
In this portion of the lab, you are using enzymes to cut the DNA at specific nucleotide sequences in
order to make fragments. Different individuals’ DNA will be cut in different places, giving them a
1. Each team will need 5 reaction tubes labeled 1, 2, 3, CS and UR (for suspects 1, 2, & 3; cells from
suit, and un restricted reference DNA).
2. Obtain DNA, enzyme and 2X restriction buffer from stations in the classroom.
•Change tips with each reagent so as not to contaminate stocks.
•Keep reagents on ice to slow the action of nonspecific DNAses.
•Always run a balanced centrifuge.
3. In each tube, combine three reagents:
4 µL of enzyme (remember: The UR tube gets water instead of enzyme!)
6 µL of 2X restriction buffer (2X RB)
2 µL of respective DNA
4. After all three reagents have been added to the tubes, close their caps tightly and mix them by
giving them a 2-3 sec. spin in a microcentrifuge. (Be sure microcentrifuge rotor is BALANCED
5. Place the tubes in a 37°C water bath for 30-45 minutes. Your teacher will store them in the
D A Y 2. Load and run gels.
In this portion of the lab, you are using electrophoresis to pull the negatively charged DNA out of the
wells and down the gel. Smaller fragments will move further than larger fragments.
6. Obtain your DNA samples.
7. Your teacher will demonstrate how to pour your gel. Put 25ml of hot agarose into a measuring
tube. Set up the casting tray so that the gates are up and the screws are tight. Put the comb in
place. Pour the agarose into the tray and let it solidify.
8. When the gel is solidified, set the casting tray with gel in the electrophoresis box with 300 ml 1X
TAE buffer. (Be sure to lower and secure the gates!) At what end of the gel should the wells be?
9. To each of your five DNA sample tubes, add 2 µL loading dye. Give them all a quick spin in the
microcentrifuge to mix in the dye.
10. Load 12 µL of each sample into a separate well in the gel. Load them in the order 1, 2, 3, CS, U.
Be sure you indicate in your notes which well contains which DNA!!! (draw a picture of gel and
11. Turn the power supply ON and set to about 100 volts.
12. Turn OFF the power supply when loading dye has reached about 1 cm from the end of the gel (about
45 minutes). Remove casting tray. Gently lift gel to plastic weigh boat and put into your ziploc
baggie (w/o buffer). These will be stored in the refrigerator overnight.
D A Y 3. Stain, photograph (done for you) and analyze (you do) gels.
The DNA has moved down the gel, but you can’t see it. We need to use a dye that will interact
with/stain the DNA (Sybr Green). After the DNA has been stained, we can see the bands it forms under
UV light. You will not do these steps yourself, but you will receive a photo of the gel to analyze.
• Use caution with the Sybr Green DNA stain. Notify the teacher if you spill some.
•UV light can damage unprotected eyes and skin. Never look directly into an unshielded UV light
source. The transilluminator is safe, since it will not turn on unless the plastic safety shield
is lowered over the gel.
13. Carefully slide your gel into a staining tray.
14. Your teacher will add the DNA staining solution called Sybr Green.
15. After 10-15-10, remove your gel to your weigh boat and take it to the Documentation Station.
16. Place your gel on the surface of the transilluminator. When the safety-lid is closed, this "box"
emits ultraviolet light that makes SybrGreen-coated DNA fragments glow. Look at your gels!
1 7 . Take a photograph of your gel. After 1 minute of developing time, peel the backing away to
separate the print from the negative. Do not get the sticky stuff on you or your
Attach your photo and label each sample in each well.
Conclusion Questions – Incorporate the answers to these questions into your
1. Into how many pieces are the DNA samples cut?
2. At which end of the gel are the fragments smallest?
3. What are restriction enzymes and how do scientists (and you!) use them as "tools"?
4. Why do we use restriction buffer in the reaction mix?
5. What is the purpose of using a "loading dye"?
6. What kind of pattern on a gel does uncut DNA leave?
7. Since you cannot "see" the tiny bit of DNA in the reaction tubes or on the gel while it is running,
what "trick" is used to visualize the DNA after electrophoresis is completed?
8. Approximately how many of each DNA fragment are in each band that you see on the gel?
9. What did you determine from the gel – i.e. “who stole the cookie from the cookie jar?”
10. What societal and ethical issues are raised by the application of DNA fingerprinting?
11. Are there uses for DNA fingerprinting that could be detrimental or discriminating to the average
12. Should DNA fingerprinting be mandatory for all people? Why or why not?
13. When this method of DNA fingerprinting is used on real human DNA what is different than the
technique we did?