Biogen Idec Community Laboratory
Our Genes, Our Selves
DNA Testing: Should Your Patient Take the Medicine?
Introduction:
Genes are sections of DNA passed from parent to offspring which are sorted
randomly during reproduction to determine an organism’s traits. One gene codes
for one protein. If a gene is faulty and produces a bad protein or does not
produce it at all, genetic disorders can result.
There are many different kinds of cancer, including Leukemia, a type of blood
cancer. One new therapy (medicine) for patients with Leukemia is CancerKiller.
However, CancerKiller has been shown to be harmful to people who have a
specific genetic disorder. This genetic disorder makes some people unable to
correctly produce a special enzyme, called TPMT, in their bodies. If people who
make fTPMT (faulty TPMT) in their bodies take CancerKiller, it will have very
harmful side effects.
In this lab, we will test samples from patients to see if they produce the working
TPMT enzyme. Because CancerKiller can be harmful to people who produce
fTPMT, it is important for us to test them to see if they produce TPMT or fTPMT
before they can take the medicine. Patients who make fTPMT cannot take the new
medicine.
Purpose: Is it safe for the patient you are testing to take CancerKiller?
Materials:
1.5 mL tubes and rack
Picofuge
p10 Micropipet and tips
p20 Micropipet and tips
Gel boxes
Power supplies
Pre-cast 1% agarose gels
UV light box/camera
Heat block
Tupperware containers
Paper towels
Adapted from Community Lab, Cambridge, MA
Tubes labeled ():
Patient samples (PT#)
+control samples (TPMT)
-control samples (fTPMT)
Buffer
Water
Restriction EnzymesHind III and Nhe1
DNA Standard Ladder (Ladder)
Sample Loading Buffer(SB)
draft 10/10/05
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Biogen Idec Community Laboratory
Setting Up the Restriction Digest:
A restriction digest is like a pair of molecular “scissors”. These scissors cut the DNA
strand into pieces in certain places. In this case, the restriction digest will cut the
DNA where the working TPMT enzyme gene should be so that we can see if the patient
has the working enzyme (TPMT) or the genetically disordered faulty enzyme (fTPMT).
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1. Obtain a Patient Sample (PT#), which contains the patient’s DNA.
2. Write your patient number on the blank below.
3. Obtain one of the Control Samples, either TPMT (working enzyme)DNA
or the fTPMT (faulty enzyme) DNA. Circle the control that you received
below.
4. Label both tubes (patient sample and control sample) with your initials.
5. Your bench mates will each also have a patient sample and a control
sample. Between the four of you, there will be:
 4 patient samples
 2 control samples with TPMT
 2 control samples with fTPMT
6. Add the following to each tube:
HINT: Use the P-20 micropipet, set to 14 µl, for the water. Use the P-10
micropipet for the buffers and restriction enzymes (Hind III and Nhe 1).
Change tips every time you use the micropipet!
Patient #
__________
14µl Water
2 µl Buffer
1 µl Hind III
1 µl Nhe 1
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Control: TPMT / fTPMT
(circle one)
14 µl Water
2 µl Buffer
1 µl Hind III
1 µl Nhe 1
7. Spin your tubes briefly in a picofuge for 5 seconds to ensure that all liquid
is at the bottom of the tube. Make sure to balance your tubes!
8. Put your tubes in the 37° heat block for 20 minutes.
9. Record your start and stop time.
Adapted from Community Lab, Cambridge, MA
Start: ____________
Stop: _____________
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Biogen Idec Community Laboratory
Gel Electrophoresis:
To see if your patient has the faulty enzyme (fTPMT), you will be performing a
technique called Gel Electrophoresis. Gel electrophoresis is used to separate DNA or
fragments based on size. To do this we put the DNA into a gel made from agarose
and run electricity through it. The agarose gel allows smaller (shorter) fragments of
DNA to travel more quickly than longer fragments of DNA.
You will be using agarose gels that have already been put into the gel electrophoresis
tray. While your DNA samples are on the heat block, your instructor will review the
steps taken to make these gels and set them up in the boxes.
Here is a picture of a gel electrophoresis box:
Power Supply
Leads
Super
Safe Lid
Gel Tray
Buffer Chamber
Double
Sided
Comb (2)
Adapted from Community Lab, Cambridge, MA
draft 10/10/05
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Biogen Idec Community Laboratory
Loading and Running the Gel:
NOTE: It is always important to wear gloves while in the laboratory. It is especially
important when you are handling materials, such as agarose gels. If you need to
touch the gel, first check with the instructor.
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1. Remove DNA samples from the heat block.
2. Spin samples for 5 seconds in picofuge to remove condensation.
3. Using the P-10 micropipet, add 2 uL of sample loading buffer (SB) to each of
your tubes. SB gives weight to the samples so they drop evenly into the wells.
SB also contains a tracking dye to monitor the gel while it’s running.
4. Gently tap bottom of tube to mix sample
5. Spin samples for 5 seconds in picofuge.
6. Designate one person in your group to use the P-20 micropipet to load 20
uL of DNA standard ladder (Ladder) to one of the outside wells on the gel.
HINT: To load the samples onto the gel, carefully insert micropipet tip
until the tip is just below the top of the well. Be careful not to stick the
pipet tip through the well. (see diagram below)
Carefully fill the well by slowly depressing the micropipet
control button until it reaches the first “stop”. Try not to push the
micropipet control button further because it will introduce air bubbles
into the well. The air bubbles may cause your sample to leak out.
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7. Have another person load 20 uL of DNA standard ladder to the other
outside well.
NOTE:You will use the DNA standard ladder wells to compare the sizes of
the patient and control samples.
Adapted from Community Lab, Cambridge, MA
draft 10/10/05
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Biogen Idec Community Laboratory
Now you and each of your bench mates will take turns loading their patient samples
and control samples into the gel by following the steps below:
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8. Using the P-20 micropipet, load 20 uL of the patient sample into a well of
your gel.
9. Make a record of which well you loaded the patient sample into on
the diagram on page 5.
10. Using the P-20 micropipet, load 20 uL of the control sample (either TPMT
or fTPMT) into a well of your gel.
11. Make a record of which well you loaded the control sample into on
the diagram below.
Black
Red
DNA ladder
DNA ladder
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12. Turn the box so ‘OWL’ is facing you. Wells are now on the left side.
13. Carefully slide the lid with attached power supply leads onto
the gel electrophoresis box. (Black anode cord is on the left. Red is on the
right.)
14. Plug the other end of the power supply leads into the power supply
box to complete the circuit.(Black to Black; Red to Red)
15. Make sure that the power supply box is set to 100 volts.
16. Make sure lid is properly placed on the box. DNA moves toward the red
cathode lead.
Adapted from Community Lab, Cambridge, MA
draft 10/10/05
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Biogen Idec Community Laboratory
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17. Turn on the power. Press RUN. Green light will come on.
NOTE: After you have turned on the gel, make sure that bubbles are
coming from the wire electrodes near the bottom of the buffer chamber,
indicating that current is in the gel.
18. Record the time that the gel started running. Start: ________________
BREAK
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19. Turn off the power by pressing RUN again. Green light will go off.
Display will say “OFF”. Record the time.
Stop: _________________
20. Remove the electrodes from the power supply box.
21. Carefully slide the lid off the unit.
22. Put paper towel in Tupperware container. Remove the gel tray from the
box. (Slightly tip tray to drain some of the extra buffer solution). Place
tray in Tupperware container to carry to the UV light box. Use caution so
the gel doesn’t slip off the tray.
 23.
An instructor will help you to place the gel tray on the UV light box and
take a picture of your gel.
 24. Let the picture develop for 1 minute then carefully peel the picture away
from the development sheet.
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Throw the development sheet away being careful not to touch the paste.
Cut out and tape the picture below.
Gel Analysis:
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1. Label the wells on your picture as you did on your gel diagram on
page 5.
2. The well with the DNA standard ladder should look like this:
Tape your picture here.
Adapted from Community Lab, Cambridge, MA
draft 10/10/05
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Biogen Idec Community Laboratory
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3. Compare the size of the bands in your patient sample to the DNA ladder.
4. Estimate the band size(s) of your patient sample: _____________________
5. Compare the size of the bands in your control sample to the DNA ladder.
6. Estimate the band size(s) of your control sample: _____________________
7. Partner’s patient sample band size____________________
8. Clean up!! Throw your tubes away in the yellow waste bin. Place
micropipets back on the rack.
9. Discuss with your partner if your patient should take the cancer medicine.
10. Record on the class data table your final analysis of whether or not your
patient should take the medicine.
Analysis questions:
1. Should your patient take the cancer medicine? Why or Why not? Use
evidence from your experiment to answer the question.
Hint: The DNA of patients who have the working enzyme gene, TPMT, will have been digested by
the restriction enzymes, Hind III and Nhe1.
2. How certain are you of whether or not your patient should take the medicine?
If the side effects of a patient taking CancerKiller with faulty TPMT were lethal, do
you think the test that you ran would be sufficient to determine if your patient
should take the medicine? Explain your answer.
3. From a business point of view, do you think that there are enough patients
who are able to take CancerKiller to mass produce and market this medicine?
Use specific evidence from the class data to answer your question.
Adapted from Community Lab, Cambridge, MA
draft 10/10/05
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Biogen Idec Community Laboratory
Extended Equipment Questions:
1. What problems might arise if the agarose wasn't dissolved completely or if
there were air bubbles in the gel?
2. What causes the DNA to travel through the gel?
3. Why do smaller DNA fragments travel further down the gel?
4. A group found that the TMPT control sample formed a large band very close to
the well and did not travel through the agarose. What could have been the
source of this error?
Adapted from Community Lab, Cambridge, MA
draft 10/10/05
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