Lab # _____: Testing for HD – Gel Electrophoresis Lab Lab skills: 1, 2, 4, 5, 13, 14, 17, 18, 21, 27, 28, 29 Background: What is HD? Huntington Disease (HD) is a devastating, degenerative brain disorder for which there is, at present, no effective treatment or cure. HD slowly diminishes the affected individual’s ability to walk, think, talk and reason. Eventually, the person with HD becomes totally dependent upon others for his or her care. Huntington Disease profoundly affects the lives of entire families: emotionally, socially and economically. Named for Dr. George Huntington, who first described this hereditary disorder in 1872, HD is now recognized as one of the more common genetic disorders. More than a quarter of a million Americans have HD or are “at risk” of inheriting the disease from an affected parent. HD affects as many people as Hemophilia, Cystic Fibrosis or muscular dystrophy. Early symptoms of Huntington Disease may affect cognitive ability or mobility and include depression, mood swings, forgetfulness, clumsiness, involuntary twitching and lack of coordination. As the disease progresses, concentration and short-term memory diminish and involuntary movements of the head, trunk and limbs increase. Walking, speaking and swallowing abilities deteriorate. Eventually the person is unable to care for him or herself. Death follows from complications such as choking, infection or heart failure. HD typically begins in mid-life, between the ages of 30 and 45, though onset may occur as early as the age of 2. Children who develop the juvenile form of the disease rarely live to adulthood. HD affects males and females equally and crosses all ethnic and racial boundaries. Each child of a person with HD has a 50-50 chance of inheriting the fatal gene. Everyone who carries the gene will develop the disease. In 1993, the HD gene was isolated and a direct genetic test developed which can accurately determine whether a person carries the HD gene. The test cannot predict when symptoms will begin. However, in the absence of a cure, some individuals “at risk” elect not to take the test. How does the disease get inherited? Each of us has 46 chromosomes which come in pairs, one member of the pair comes from each parent. Therefore, 23 chromosomes are from the mother, and 23 chromosomes are from the father. There are two types of chromosomes: 1) autosomal chromosomes, which are the first 22 pairs, and 2) sex chromosomes, which are the 23rd pair (the 23rd pair in females consists of two X-chromosomes, and the 23rd pair in males consists of an X-chromosome and a Y-chromosome). Since Huntington Disease is autosomal dominant, this means the gene involved is on an autosomal chromosome (not one of the sex chromosomes). It has recently been localized on the fourth autosomal chromosome pair (the #4 chromosome). In affected individuals, one gene of this gene pair (the HD gene) is not functioning correctly and expresses itself more strongly, or 'dominates', the other working gene. Since it is not on one of the sex chromosomes, it can affect both males and females. Pedigree chart: An affected parent passes either the HD gene, or the other working gene, to their offspring. There is a 50% (1 in 2) chance at each pregnancy that a child of an affected parent will receive the gene for HD. The age of onset, degree and type of symptoms, as well as rate of progression varies from person to person. Hypothesis: We think that genetic testing can be used to determine if someone has a genetic disease. Testing for HD ~ Gel Electrophoresis Lab 1 Genetic Testing Laboratory Procedure: A. Prepare agarose solution: 1. Place 1.8 grams (1/2 teaspoon) of agarose and 70 ml of electrophoresis buffer into a flask or bottle. 2. Follow your teacher’s instructions to heat the agarose solution until it has dissolved completely. 3. Insert a thermometer into the agarose solution. Allow the solution to cool to 60 oC. While the agarose solution cools, continue with the following steps. B. Set up the electrophoresis apparatus: 4. Remove the gel box lid by pulling it up vertically. 5. Place the gel casting tray on the raised platform in center of gel box. The tray can only fit properly in one direction. 6. Place the white, wedge-shaped dams in the appropriate slots at either end of gel casting tray. The flat sides of the dams should face the gel casting tray. Be certain that the colored dots are on the same side as the matching color electrode. side view 7. Place the comb in the slots located at the end of the gel casting tray closest to the black electrode. C. Pour the gel: 8. When the agarose solution has cooled to 60oC, pour it into the gel casting tray between the two dams. The agarose solution should come ½ to ¾ of the way up the teeth of the comb. 9. Do not move or touch the gel while it cools and hardens. The gel should harden in 20 minutes. top view D. Practice loading samples: 10. While the gel hardens, each member of the lab group should practice using a micropipettor to load samples of dye into the wells on the small practice gel. 11. Use the sample in the orange tube labeled P. Follow the instructions on the Practice Gel Loading Instructions sheet. E. Remove the dams and comb: 12. Once the gel has completely hardened, remove the wedgeshaped dams by lifting them vertically out of their slots. 13. Store the dams in the spaces at the ends of the gel box. Match the colors on the gel box with the colors on the dams. 14. Gently but firmly remove the comb from the gel by pulling up with one hand, while holding the gel casting tray in place with the other hand. Set the comb aside in a safe place. Testing for HD ~ Gel Electrophoresis Lab 2 F. Load the samples to be tested: 15. Load the samples from the colored sample tubes (1 through 5) into the wells on the gel as shown below. To open the sample tube, hold the tube tightly and carefully push upward on the top tab. 16. Make sure to use a fresh disposable pipette tip to load each sample. 17. Take turns so that each member of your lab group has an opportunity to load samples into 2 or 3 wells. Key to samples: Sample Number 1 Tube Color yellow Sample 2 blue 3 pink 4 purple Sample from Jeremy Lanahan 5 aqua Sample from James Lanahan (Dad) Negative control from an unaffected individual Positive control from an individual with HD Sample from Jenny Lanahan G. Add buffer: 18. Position the gel box so that the electrode wires will reach the power supply. 19. The gel box should not be moved once you have added buffer. 20. If it is moved after the buffer is added, the samples may spill out of the wells. 21. Pour approximately 350 ml of electrophoresis buffer into the chambers at the ends of the gel. Pour slowly so that you do not wash the samples out of the wells. 22. Make sure that the buffer completely covers the surface of the gel to a depth of approximately 5 mm. Add more buffer if needed. H. Set up to run the gel: 23. Place the lid on the gel box, making sure the black and red colors match. 24. Plug electrode wires into the power source, making sure that the red and black colors match. 25. When all teams sharing a power supply have completed the previous steps, have your teacher check your work. Testing for HD ~ Gel Electrophoresis Lab 3 26. Under your teacher’s supervision you can then: Plug the power cord into the back of the power supply and into a wall socket. Turn on power switch in the back of the power supply. Use CONSTANT button to select “V” for constant voltage. Use the DISPLAY button to select “V” display for voltage. Use the +/- controls to set the voltage at 150 Volts. Press the “Start” button to start the electrophoresis process. Observe the ends of the gel box. You should be able to see the small bubbles forming on the wires in the chambers covered by buffer on the bottom of the gel box. After 5 minutes, you should be able to observe the colored samples moving through the gel. I. Observe the results: After approximately 10-15 minutes, when the bands have clearly separated, call your teacher. Your teacher will turn off the current. After your teacher has turned off the power supply, unplug the gel box from the power supply. Carefully remove the lid from the gel box. Lift the gel casting tray out of gel box. The gel is slippery, so keep the tray level and gently hold the gel to keep it from sliding out of the gel tray. Place the gel tray onto a light background surface (such as a piece of white paper). Record your results. Draw the position of the bands in each of the lanes of the gel using the diagram in the Genetic Testing Analysis section of your lab report (top of p. 6). J. Clean and store the equipment: Place the gels into the recycling container provided by your teacher. Empty the electrophoresis buffer into the recycling container provided by your teacher. Rinse and dry the gel box, gel casting tray, and comb. Place the gel casting tray and comb inside the gel box. Put the lid on the gel box. Testing for HD ~ Gel Electrophoresis Lab 4 Genetic Testing Analysis: 1. Observe and then draw the position of the bands in each of the lanes of your gel on your answer sheet to create a diagram of the gel with results. Set it up like the one shown below but also include the bands observed. 1 2 3 4 5 1 2 3 4 5 Lane 1 – Negative control 2 – Positive control 3 – Jenny 4 – Jeremy 5 – James (Dad) 1 – Negative control 2 – Positive control 3 – Jenny 4 – Jeremy 5 – James (Dad) 2. Each person inherits one copy of the huntingtin gene from each parent. Explain why the negative control sample (from an unaffected individual) only produced one band. 3. Explain why the positive control sample (from an individual affected by HD) produced two bands. 4. Which band in the positive control lane (the one closest to the well or the one farthest from the well) represents the DNA from an abnormal huntingtin gene with CAG repeats? Explain your answer. 5. Does Jenny have a gene for HD? Support your answer using the results of the gel electrophoresis. 6. Does Jeremy have a gene for HD? Support your answer using the results of the gel electrophoresis. 7. Does James (their father) have a gene for HD? Support your answer using the results of the gel electrophoresis. 8. State two ways to improve the accuracy and/or reliability of these gene tests. 9. Complete the gene testing report for James Lanahan (their father). Refer to your notes for a summary of the testing procedure. In the test results section you should state if James has HD or not. In the analysis/interpretation of results section you should identify a limitation of genetic testing for this diseaseexplain that if results are positive it is unknown as to when the effects of HD will appear. Testing for HD ~ Gel Electrophoresis Lab 5 Laboratory Report Huntington Disease Gene Testing Lab technicians (group members): ____________________________________ __________________________________ ____________________________________ __________________________________ Patient’s name: ____James Lanahan____ Date of Test: ________________________ Description of testing procedure: __________________________________________________________________________ __________________________________________________________________________ __________________________________________________________________________ __________________________________________________________________________ __________________________________________________________________________ __________________________________________________________________________ __________________________________________________________________________ __________________________________________________________________________ Test Results: __________________________________________________________________________ __________________________________________________________________________ __________________________________________________________________________ __________________________________________________________________________ Analysis/interpretation of test results: __________________________________________________________________________ __________________________________________________________________________ __________________________________________________________________________ __________________________________________________________________________ __________________________________________________________________________ Signature: __________________________________ Date: _________________________ Testing for HD ~ Gel Electrophoresis Lab 6 Situation: Jenny and Jeremy are the teen-aged children of James Lanahan. They have been upset because their grandmother (James’s mother) has passed away after suffering from Huntington Disease for many years. They are worried about their own futures because they know that HD can be passed on and that it could be years before it shows up or it could be right away. James is concerned because he has been experiencing muscle spasms in his neck, which could be an early onset symptom of HD. The family has decided to have genetic testing. They all went together so that they could support each other if they found out any bad news. Remember, there is a 50-50 chance that the gene is passed on to the children of an affected individual. If the father is negative that means he does not carry the gene so the children do not need to worry. Narrator: Jenny, Jeremy, and their father have decided to undergo genetic testing for Huntington Disease. A nurse is about to take their blood sample… Nurse: I see that each of you met with a genetic counselor and signed the required informed consent forms. Now I’m going to draw a blood sample from each of you. Jenny, I’ll take you first. Jenny: Wait, I have a question: how do you test my blood for the gene? Nurse: We don’t do the testing here at this facility. We send your blood to the Pulaski High School genetics lab in Room 211 that specializes in HD testing. Jeremy: What does the lab do with my blood? Nurse: A lab technician spins your blood to get a layer of white blood cells. Then they take some of those white blood cells and isolate the DNA from them. Jeremy: How do they look at the DNA to find the genes? Nurse: You can’t see genes, even with a microscope. The lab has to make many copies of your DNA just to run tests. This process is called the polymerase chain reaction, or PCR. Only a small part of your genome containing the huntingtin gene will be copied. Jeremy: Once they get those gene copies, how can they tell if the genes are mutated or different from normal? Nurse: Gel electrophoresis is used to study DNA. The gene copies are put in an agarose gel, which looks like a piece of Jell-O. The gel is placed in a box filled with liquid that will carry an electric current. When the current is on, the gene copies will move with the current to certain places in the gel, depending on how big they are. Jenny: So how do they tell if we have the mutated gene? Nurse: Since the Huntington Disease gene has extra nucleotides, called CAG repeats, it is longer than the normal gene. If you don’t have the HD gene, you have normal length DNA pieces. On the gel, the normal length pieces of DNA move to a certain spot. The longer pieces move to a different spot. The lab technician can tell if you have a normal gene or a mutated gene by looking where the DNA pieces end up on the gel. Dad: Seems like there are lots of steps to this process. There are lots of places where something could go wrong. Is the test really accurate? Will it tell for sure whether we have the gene? Narrator: Tomorrow, your lab group will act as laboratory technicians. You will use gel electrophoresis to analyze the simulated DNA samples from Jenny, Jeremy, and Dad. You will then analyze the results to determine if the genes are normal or mutated. Testing for HD ~ Gel Electrophoresis Lab 7 Testing for HD ~ Gel Electrophoresis Lab 8