Where’s the “CAT”? A DNA Fingerprinting/Gel Electrophoresis Simulation DNA fingerprinting (profiling) is currently being used in forensic cases to assess the probability of a suspect’s involvement in certain crimes. DNA may be extracted from relatively small samples of cells such as a blood stain or semen stain. When performed under properly controlled conditions and interpreted by an expert forensic scientist, such profiling can link a suspect to a particular incident with compelling accuracy. DNA profiling can also be used to determine the parents of a child. As a forensic scientist, this is the situation you have been called upon to solve. Two families, the Jacobsons and the Olsons had babies at the same time at the same hospital. The Jacobsons think that the babies were switched. Electrophoresis can be used to help settle the matter. DNA profiling works in the following manner. 1.Cells are taken from an individual. The cells are broken open and the DNA is extracted. 2..The DNA is chemically split into individual strands by restriction enzymes. DNA restriction enzymes cut the DNA into smaller pieces. These enzymes only cut the DNA at specific places based upon specific sequences of nucleotides. Theses fragments of DNA (known as RFLPs –Restriction Fragment Length Polymorphism) are placed into wells of an electrophoretic gel and the different sized pieces are separated using a process called gel electrophoresis. The strands are separated during electrophoresis by an electrical current. DNA has a negative charge and is pulled toward the positive end of the gel. The DNA fragments are initially placed into pools at the negative end of the gel. The largest strands move the slowest and the shortest strands move the fastest. After the DNA has been separated into its bands, a “probe” (radioactive group of three nucleotides) is added to the DNA strands. It will only bond with the complementary nucleotides on the DNA. Once the probes are added, a piece of film is placed over the DNA bands. The radioactive particles cause lines to develop on the film. (This step will not be done in the simulation, nor in the “real” lab experience.) Each person produces a different banding pattern on the film because they have inherited different nucleotide sequences. In this, your first D NA fingerprinting case, you will simulate the DNA profiling of the woman, husband, child, and suspect. From these tests you will determine the biological father of the child. Procedure Materials (per group) Paper base sequences for standard (control),Baby, Anderson’s, Olsons DNA probes and highlighter Butcher Block Paper It might be helpful if you first determine who will be responsible for which person’s DNA. 1. Cut out all of the DNA strands. On the back of each strand write the name of the person to whom it belongs (ie. “Mrs. Olson”) 2. Tape together the strips of your samples by covering the subscripts in order to get one long paper strip of bases for each person. 3. Start with the Standard DNA. Scan the strip until you find the sequence “GGCC”. Using your scissors to act as a restriction enzyme, cut across that strip between the center G and C. You will be forming a fragment that ends with GG and another that begins with CC. (HINT: each sample should have 5 of these sites, giving 6 fragments, with the exception of the standard DNA.) Count the number of bases in each fragment and write that number on the back of the fragment. 4. Obtain a piece of poster paper. Label the lanes by writing the titles of the different samples across the top of the paper. Follow the guidelines offered by your teacher. 5. Place the fragments on the simulated electrophoresis gel sheet so that the bands will represent the final result of the process. Remember that the larger strands move more slowly. Separate the DNA strands into proper sequence. Do this first for the standard and then for the other people in question. Leave a 1-cm space between the different sized strands for the standard DNA. The standard’s fragments serve as a guide for the placement of the other fragments. 6. Tape the DNA fragment strands in place using clear tape. Tape each fragment in its proper position under the appropriate heading (standard fragments in the lane under the “STANDARD” heading.) NOTE: When placing the DNA strands in proper location on the electrophoresis simulated gel, you must place the same length DNA strand the same distance from the beginning point if it is the same length. Odd numbered nucleotide lengths will move to positions between the even numbered ones. 7. Using scotch tape, attach each DNA probe (GTA) to the proper locations on the DNA strands. Remember, you are matching complementary bases. Highlight where they are with a yellow highlighter. 8. After radioactively labeling the DNA segments with the probes, compare the banding pattern of each person to that of the standard and to each other to determine the biological father of the child. Draw the banding pattern (where the probes are) of your gel here: Answer the questions on the next page. Name_________________________ DATE____________ Questions for Where’s the CAT activity 1. By what 2 properties does electrophoresis separate DNA? 2. From where can DNA for a DNA profile be extracted? 3. What is the purpose of restriction enzyme? 4. When looking at the results of an electrophoresis gel, how do you know which DNA fragments were short and which were long? 5. Why did the probes adhere to some fragments and not others? 6. Why are the probes radioactive? 7. Before adding the probes, could you tell who the father of the baby was? Explain. 8. Who is the father of the baby? Explain how you made your conclusion. 9. If you were to do DNA profiling for a criminal case rather than a paternity case, how would your analysis of results be different? 10. Conclude what you learned from this lab by writing a paragraph. Include the following underlined terms. DNA, fragments, size, charge, negative, positive, small, large, probe, complementary, restriction enzyme, sequence, compare. Use the back of this sheet.