Activity 1.3.1: DNA Detectives
Introduction
Bones can provide a snapshot of the identity of a person- they can predict height,
stature, gender, ethnicity and even age. However, it is what lies inside these hard
calcified tissues, the DNA housed inside the body’s cells, that holds the key to true
genetic identity. Tissue is made up of many cells, the building blocks of life. Tucked
inside the body’s cells, you will find chromosomes. These structures house your
genes and contain the DNA code necessary for the production of all of the proteins
that keep your body functioning. Your DNA provides a unique code of over three
billion base pairs. Unless you are an identical twin, there is no other person on the
planet with your same code. And although only one tenth of one percent of this DNA
differs from person to person (that’s still 3 million base pairs!), the regions that vary
provide a true genetic blueprint of an individual. This amazing molecule is tiny -invisible to the naked eye -- but it is often the only key that can link killers to a crime,
parents to their children or a person to his/her own bones.
In PBS, you learned about the molecular biology techniques that allow scientists to
explore our DNA. PCR, Polymerase Chain Reaction, is the copy machine; the
revolutionary process that allows scientists to replicate even the tiniest speck of
DNA. Restriction endonucleases (enzymes) are the molecular scissors that can cut
DNA in specific locations. Your specific code determines the number of times this
set of scissors will snip and the number and size of DNA pieces that will be left
behind. These pieces can then be separated and compared using the process of gel
electrophoresis. As these fragments move, their varying lengths propel them through
the gel at different speeds. Scientists can use these RFLPs, Restriction Fragment
Length Polymorphisms, a set of DNA puzzle pieces unique to only you, to create a
pattern called a DNA fingerprint. Similar to the unique fingerprint from your hands,
this DNA fingerprint provides key information about human identity and, at the
smallest level, provides a clue to what makes you, you.
In Lesson 2, you used basic forensic anthropology to analyze bones and to provide
a preliminary snapshot of the two individuals found in the park. Using the clues you
have unearthed, the local police force has run these descriptions through their
missing person files. Two people fitting the description for each skeleton have been
reported missing in the past year. You will now work as a forensic DNA analyst to
evaluate DNA samples found in the bones of the skeletons and compare each
unique DNA fingerprint to the genetic material of the people who have gone missing.
In this activity, you will explore how restriction enzymes work and how they allow us
to visualize differences in our DNA. You will use these molecular scissors to cut the
DNA in each sample, and you will use gel electrophoresis to analyze the pattern of
bands that are left behind. DNA work takes care and precision. Work carefully to
identify these individuals and finally give their families some peace.
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Human Body Systems Activity 1.3.1 DNA Detectives – Page 1
Equipment
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Computer with Internet access
Laboratory journal
Edvotek DNA Fingerprinting II Kit
o Predigested DNA from bone
o DNA samples from missing persons
o Restriction enzymes (EcoRI and HindIII)
o Reaction buffer
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Microcentrifuge tubes
37°C water bath
One agarose gel (0.8% agarose; 8 wells)
Tris-Acetate-EDTA (TAE) gel electrophoresis buffer
Gel electrophoresis apparatus
Micropipettor (20 µl)
Disposable micropipette tips
Light box
Safety goggles
Lab apron
Activity 1.3.1 Student Resource Sheet
Procedure
In this activity, you will continue work on the skeleton your team analyzed in Lesson
2. You will compare the DNA found inside of the bones of your skeleton to the two
individuals who match the profile you have provided. DNA samples provided by the
family of those who have gone missing will each be cut, or digested, with two
restriction enzymes in separate reactions and will be compared to DNA isolated from
the humerus of the unearthed skeleton. The DNA extracted from your skeleton has
already been digested with these same two enzymes.
PART I: DNA DIGESTION WITH RESTRICTION ENZYMES
1. Analyze DNA from the two individuals who have been linked to your skeleton:
Missing Person #1 and Missing Person #2.
2. Obtain four, clean microcentrifuge tubes. All other reagents should be located at
your lab station.
3. Label microcentrifuge tubes 1-4 for four restriction enzyme digestion reactions.
Make sure to label each tube with the name of your group. Also, make sure to
note the contents of each tube in your laboratory journal. You will process four
DNA samples:
o Tube 1- Missing Person #1 cut with Enzyme 1
o Tube 2- Missing Person #1 cut with Enzyme 2
o Tube 3- Missing Person #2 cut with Enzyme 1
o Tube 4- Missing Person #2 cut with Enzyme 2
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4. Use the micropipettor to dispense 10µl of Enzyme Reaction Buffer into each of
the four labeled tubes.
5. Add DNA and enzymes to tubes 1-4 according to the following information. Each
tube will have a final volume of 40µl. Remember to use a fresh pipette tip for
each transfer. You do not want to contaminate your samples.
o Tube 1- 15µl DNA 1 + 15µl Enzyme 1
o Tube 2- 15µl DNA 1 + 15µl Enzyme 2
o Tube 3- 15µl DNA 2 + 15µl Enzyme 1
o Tube 4- 15µl DNA 2 + 15µl Enzyme 2
6. Cap each tube and gently tap the side of each tube to mix. Tap the tube gently
on the desk to make sure all of the contents are in the bottom of the tube.
7. Incubate your tubes in a 37°C water bath for 45-60 minutes. Place your tubes in
the bath as directed by your teacher. At the end of the incubation time, you will
need to add 5µl of 10X gel loading solution to tubes 1-4 to stop the reaction.
Depending on time, your teacher may complete this step for you. As your
samples digest, continue with Steps 8 -10.
8. Obtain a Student Resource Sheet from your teacher.
9. With your partner, review the science behind restriction enzymes. Use the
websites listed below to complete the questions and activities in Part A of the
Student Response Sheet.
o Dolan DNA Learning Center: Restriction Enzymes
http://www.dnalc.org/ddnalc/resources/restriction.html
o Access Excellence Classic Collection: Restriction Enzymes
Background Paper
http://www.accessexcellence.org/AE/AEC/CC/restriction.php
10. Review the process of gel electrophoresis by viewing the animations listed
below. Use information from the sites to complete the activity described in Part B
of the Student Response Sheet.
o Genetic Science Learning Center – Gel Electrophoresis Virtual Lab
http://learn.genetics.utah.edu/content/labs/gel/
o DNA Interactive: Gel Electrophoresis
http://www.dnai.org/text/mediashowcase/index2.html?id=1014
PART II: GEL ELECTROPHORESIS OF RESTRICTION FRAGMENTS
11. If you are making the gel, follow your teacher’s instructions to melt and pour the
agarose.
12. Remove the comb carefully from the agarose gel and place the gel in the
electrophoresis chamber.
13. Fill the chamber with TAE buffer, making sure the gel is completely submerged.
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14. Obtain your sample tubes 1-4. Gently flick the side of each tube to mix the
contents. Digested DNA samples from the bone and the standard DNA marker
will be located at each lab station or table.
15. Heat the samples, including the standard DNA marker and the DNA extracted
from bone, for two minutes at 65°C. Your teacher will direct you to the heat block
or water bath. Allow the samples to cool a few minutes before loading them on
the gel.
16. While your samples cool, practice loading samples on the practice gels.
17. When you are ready, load 40µl of each sample into the gel. Make sure to use a
fresh tip for each sample.
o Lane 1: Standard DNA Marker
o Lane 2: DNA from Bone cut with Enzyme 1
o Lane 3: DNA from Bone cut with Enzyme 2
o Lane 4: Tube 1- Missing Person 1/Enzyme 1
o Lane 5: Tube 2- Missing Person 1/Enzyme 2
o Lane 6: Tube 3- Missing Person 2/Enzyme 1
o Lane 7: Tube 4- Missing Person 2/Enzyme 2
18. Draw a diagram of the gel in your lab notebook. Make sure to clearly indicate
which sample is in which well.
19. Follow your teacher’s instructions to assemble the gel electrophoresis apparatus
and connect the power supply. Be sure to check the orientation of your gel. The
DNA containing wells should be closer to the negative pole and farther away
from the positive pole.
20. Follow the teacher’s instructions to turn on the power supply.
21. Check the DNA samples 5 minutes after turning on the power supply. Make sure
the loading dye is migrating out of the well and moving towards the positive pole.
22. Check your gel every ten minutes and turn off the power supply when the dye is
near the bottom of the gel. The gel should take 30-45 minutes to run. During this
time, continue work on the Student Resource Sheet.
23. When your gel is finished running, follow your teacher’s instructions to stain and
visualize the DNA fragments.
24. Examine your results and make a sketch of the gel in your lab notebook.
25. Analyze the pattern of fragments that you see. Do the DNA fingerprints from the
bone sample match up with either of the missing persons? Explain your findings
in your lab notebook.
26. Return to the case report created by the forensic anthropologist. Add a new
heading:
o DNA Analysis: Referring to your gel electrophoresis results, clearly
describe the findings of the DNA analysis. Make sure your
explanation is clear (and well supported by what you see in the gel)
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and provide a clear conclusion as to the identity of the skeleton in
the park. If possible, attach a labeled photograph of your gel to the
completed case report.
o Final Conclusion: Use information from the entire case to sum up
the identification experience.
27. Answer the conclusion questions.
Conclusion
1. Explain the relationship between the following words – cells, genes,
chromosomes, tissues, DNA, proteins.
2. Explain why scientists probably used PCR when they prepared the DNA for your
experiment.
3. In the space below, write a paragraph explaining what you would say to the other
family to convince them that the science techniques used prove the bones do not
belong to their loved one.
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4. Suggest reasons why it was helpful to digest each of your samples with two
different restriction enzymes? How do the results of your gel reinforce this point?
5. Explain how the code in your DNA relates to your physical appearance as well as
the functioning of your body. How might a change in this code impact the body?
6. Other than information from bone and from DNA analysis, what other
characteristics/identifiers can be used to identify this skeleton?
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Human Body Systems Activity 1.3.1 DNA Detectives – Page 6