Applications of Biotechnology Electrophoresis lab: (without the DNA) Introduction to micropipetters and electrophoresis equipment

Transcription

1 Applications of Biotechnology Electrophoresis lab: (without the DNA) Introduction to micropipetters and electrophoresis equipment Materials- Gather the following items at your table: Eight samples for electrophoresis (one for each family member, A-F, plus positive and negative controls. A 20 ul micropipettor and tipbox Electrophoresis chamber Power supply (to be shared) Electrophoresis buffer (to be shared) Activity #1 - Electrophoresis Electrophoresis is a method for separating molecules by size and charge. The samples are placed in a special gel and subjected to an electric field. Negatively-charged molecules, such as DNA fragments, move toward the positive electrode; whereas, positively-charged molecules move toward the negative electrode. The smallest fragments or molecules travel the farthest from their original position, since they are less impeded by the gel matrix. Following the electrophoresis, the bands of the sample in the gel are visualized using dyes such as ethidium bromide or by using autoradiography. The materials needed for electrophoresis are expensive and often toxic. For that reason, we ve substituted dyes and other safe chemicals for today s activity. You will run the gels and interpret the results based on a hypothetical situation involving a family affected by DMD. Diagnosis of a Genetic Disease You and your lab partners are going to play the role of laboratory technicians in a local hospital. You will perform a DNA screening test on the members of the Smith family. You will then interpret the results and decide how to handle the genetic information obtained in the test. Case Background Mary and John Smith have three children: Daniel, age 5, Alice, age 4, and Michael, age 1. Mary and John noticed that Daniel was having difficulty climbing the stairs and complained about being extremely tired after playing tag with his sister. The family doctor ordered several tests on Daniel, which confirmed he had a disease called Duchenne Muscular Dystrophy (DMD). DMD is a genetic disease that results from damage to a gene on the X-chromosome. DMD is more common among males, and they inherit the defective copy of the gene from their mother. Individuals with DMD usually appear healthy until the age of four or five, when their muscles start to weaken. The doctor told Mr. and Mrs. Smith that Daniel would probably need a wheelchair within a few years and would probably die before reaching his 21 st birthday. The Smiths became concerned about their remaining children, and wondered about their chances for developing DMD. Mr. Smith is clearly not affected by DMD, but Mrs. Smith must be a carrier for the disease; therefore, there is a chance that Michael will also be affected. Although Alice could not have DMD, she could be a carrier. Mary has just found out that she is pregnant again and is also concerned about her unborn child being affected (if male) or a carrier (if female). The doctor who diagnosed Daniel as having DMD mentioned that there are genetic screening tests available to determine whether each family member had the defective copy of the gene. After consulting with a genetic counselor, the Smiths decided they should all undergo genetic testing. A laboratory technician took a small drop of blood from the fingertip of each member

2 of the Smith family, and Mary underwent a fetal blood sampling procedure so that the genetic make-up of her unborn child could be determined. The technician sent the samples to a genetic testing lab for screening and told the Smiths to come back in three days for the results. At the genetic testing lab, a technician isolated DNA from the white blood cells of each blood sample. The isolated DNA contained all of the genes present in each individual but the lab was only interested in the gene for DMD. Therefore, the technician used a procedure called the Polymerase Chain Reaction (PCR) to make copies of ( amplify ) just the section of DNA containing the DMD gene. Once a large number of copies of the DMD gene had been obtained from each sample, the amplified samples were sent to your lab group for analysis. The defective copy of the gene that causes DMD, as well as its normal version, have been well characterized and it is known that the mutation that causes DMD is a deletion in the normal version of the gene. Due to the deleted region, the defective version of the gene will be smaller than the normal version of the gene; therefore, the two versions of the genes can be separated from each other using gel electrophoresis. Mary has two copies of the DMD gene, one normal and one defective (the defective gene has a piece of the DNA deleted from it). Because he is male, John has only one copy of the DMD gene and it is normal. Daniel also has only one copy of the DMD gene and it is defective. Knowing the genetic make-up of Mary, John, and Daniel will allow you to determine the genetic make-up of the other family members, and the unborn child, by comparing their DNA samples with those of Mary, John, and Daniel. You are going to perform an analysis on the DNA samples from the Smith family and determine the genetic profile of each family member. Problem Statement: Write a statement or questions regarding what we are trying to determine by completing this lab. Hypothesis: Use the information about Mary and John to find the probabilities of having a child with DMD. Based off on your results, make a prediction as to whether the new baby with have DMD. Directions: Setting up the Electrophoresis Chamber with pre-made gel 1. Place the gel on a plastic tray well side up and place in the electrophoresis chamber so the wells are close to the black electrode. The DNA samples will run towards the red electrode. 2. Cover the gel and fill both sides of the chamber with electrophoresis buffer. The buffer should just cover the gel. Now you are ready to load the gel. Loading the Samples 1. Use a micropipettor to load 15 l of each sample into separate wells as described below. a. Set the micropipettor to 15 l. b. Place a clean (unused) tip on the micropipettor.

3 c. Depress the plunger at the end of the micropipettor until you reach the first stop. d. Place the micropipettor tip into the sample and slowly release the plunger to withdraw sample into the tip. e. Using two hands, steady the pipette over the well. f. Dip the pipette tip through the surface of the buffer, center it over the well, and gently depress the micropipette plunger to slowly expel the sample into the appropriate well. If the tip is centered over the well, the sample will sink to the bottom of the well. Do not release your thumb until the micropipette tip is out of the gel. g. Eject the used tip into the trash and obtain a new (unused) tip before loading the next sample. 2. Label the wells in the picture of the gel below to show where you placed each sample in your gel. The gel should be loaded (and labeled) left to right as follows: Tube A (Mary), Tube B (John), Tube C (Daniel), Tube D (Alice), Tube E (Michael), Tube F (fetus), Tube G [DMD control] Tube H [normal control]. Running the Agarose Gel 1. Put the top on the electrophoresis chamber and connect the electrical leads, red to red and black to black. Make sure both electrodes are connected to one channel of the power supply. 2. Notify your teacher that you are ready to turn the unit on. 3. Set the power source on 130 volts and turn the unit on. CAUTION: Electrical shock hazard. Do NOT put fingers or other objects into the box while the power supply is on! 4. After the current is applied, you should see small bubbles rising from the electrode wires in the gel chamber. Very shortly, you should see the samples moving through the gel. 5. Run the electrophoresis for approximately 15 minutes. Check to monitor the progress of the dye front. NOTE: If you leave the gel running for too long, the samples will run off the end of the gel and get lost in the buffer. 6. When the dye has moved sufficiently through the gel to clearly see band separation, turn off the power supply. Then disconnect the leads. 7. On the picture of the gel below, draw in the bands that resulted after electrophoresis. Be sure to show each band in its proper position.

4 Complete the data table below: Note: X D = normal version of DMD gene; X d = defective version of DMD gene Tube A Tube B Tube C Tube D Tube E Tube F Mother: Mary Father: John Son: Daniel Daughter: Son: Michael Fetus: M or F? Alice Carrier Healthy Has DMD alleles: 2 X D X d alleles: 1 X D Y alleles: 1 X d Y alleles: alleles: alleles: Samples G and H are controls. Clean Up: Discard the agar gels into the trash. Discard pipette tips in the trash. Return the sample sets to the designated lab box. If you are the last class doing this experiment, discard the electrophoresis buffer solution down a drain. Rinse out the electrophoresis chambers and gel trays with WATER ONLY. Leave them overturned to dry. Be very careful with the delicate wires in the boxes--do not attempt to dry them out. Review and Application Questions: 1. Which allele (normal or defective) moved further in the gel? Why? 2. Is Alice (the daughter) a carrier?. How can you tell? 3. Does Michael have DMD?. How can you tell? 4. What sex is the Smith s unborn child?. How can you tell? 5. Does the fetus have DMD?. Is the fetus a carrier of DMD?. How can you tell? 6. Why are most individuals with DMD male? 7. Can a male be a carrier for DMD without having the disease?. Why or why not?

5 Answer Sheet for Teachers: 1. Which allele (normal or defective) moved further in the gel? defective_ Why? The gene has a deletion in it making the DNA smaller. Smaller DNA moves faster through gel. 2. Is Alice (the daughter) a carrier? Lane 4:yes How can you tell? She has 2 bands: one normal running high (longer goes slower), one defective running fast or low in gel. 3. Does Michael have DMD? No How can you tell? Lane 5. He has one band that runs high meaning his DNA is longer and is the normal version 4. What sex is the Smith s unborn child? female How can you tell? The 6 th lane has 2 bands. The DMD is located on X chromosome and females have 2 X chromosomes. 5. Does the fetus have DMD? No Is the fetus a carrier of DMD? Yes How can you tell? Two bands, one is normal (runs high, longer DNA) one is the lower DMD deleted version (shorter DNA runs farther in gel) 6. Why are most individuals with DMD male? Males only have 1 X chromosome and DMD is on the X chromosome. 7. Can a male be a carrier for DMD without having the disease? No Why or why not? With one X, you have it or you don t. Other questions that come up: males with DMD are not healthy enough to have children themselves.