In the Laboratory
A Metabolic Murder Mystery: A Case-Based Experiment for the Undergraduate Biochemistry Laboratory Jessica L. Childs-Disney,* Andrew D. Kauffmann, Shane G. Poplawski, Daniel R. Lysiak, Robert J. Stewart, Jane K. Arcadi, and Frank J. Dinan Department of Chemistry & Biochemistry, Canisius College, Buffalo, New York 14208 *
[email protected] Traditional laboratory experiments often require that a “cookbook” laboratory procedure be followed and a known result obtained. Experiments based on a real-life case study are designed to capture students' interests and to demonstrate the realworld importance of the laboratory experiment. Whereas casebased laboratory experiments are relatively uncommon, case-based classroom instruction in the sciences is widespread. The collection of science-based case studies found at the National Center for Case Study Teaching in Science (1) and the large number of hits this site receives each day attests to the increasing popularity of this approach. A recent book (2) provides excellent guidance for those seeking to learn about case teaching. The case study approach allows students to see the advantages that accrue from working in cooperative teams and the potential importance of the work they are doing in a real-world context. We try to provide our students with the minimal guidance consistent with time constraints and safety. One of our case-based experiments has been published (3) in this Journal. Summary of the Metabolic Mystery Case Study This case describes the events that occurred after Mrs. Patricia Stevens (name has been changed) brought her ill infant son, Ryan, to a hospital emergency room (4). The attending physician diagnosed the infant as having been poisoned with ethylene glycol. Ryan was removed from his mother and placed in foster care. Mrs. Stevens was only allowed to see her son while supervised. On one occasion, Mrs. Stevens was mistakenly left alone with Ryan, and shortly after this he became ill and died. Mrs. Stevens was charged with first-degree murder and held without bail. While in jail, Mrs. Stevens learned that she was pregnant and gave birth to a second son, David. Shortly after his birth, David exhibited symptoms very similar to Ryan's. Mrs. Stevens had never been left alone with David and could not have poisoned him. He was diagnosed as having methylmalonic acidemia (MMA; also known as methylmalonic aciduria), a recessive genetic disorder that has symptoms similar to those exhibited by victims of ethylene glycol poisoning (5). The prosecutor still decided to prosecute Mrs. Stevens. The trial judge ruled that David's MMA diagnosis could not be introduced as evidence. Mrs. Stevens was convicted of murder and sentenced to life in prison. Two scientists who had followed Mrs. Stevens' trial in the media thought that she may have been wrongly convicted. They obtained a sample of Ryan Stevens' blood and analyzed it for the presence of ethylene glycol. None was detected, but they did find 1110
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a high concentration of methylmalonic acid in the serum. Methylmalonic acid builds up in the bodies of MMA patients because of a missing or defective enzyme that is needed for its metabolism. When the scientists asked to be allowed to investigate the ethylene glycol content of the formula that Ryan had been consuming just prior to his death, they were told the sample “had disappeared”. The scientists deemed the quality of the laboratory's work to be “unacceptable”. This information was presented to the prosecutor who concluded that he “no longer believed the laboratory data” and now accepted that Ryan Stevens had died of MMA. All charges against Mrs. Stevens were subsequently dismissed. The Laboratory Experiment The Stevens' case serves as an introduction to polymerase chain reaction (PCR) amplification of DNA (6). An excellent resource for learning about PCR techniques has been published by Cold Spring Harbor Laboratory Press (7). The major cause of MMA is a genetic defect in the methylmalonyl-CoA mutase enzyme (MUT) that leads to increased concentrations of methylmalonic acid and other organic acids (5). These organic acids were misanalyzed as ethylene glycol in the Stevens' case. One mutation that causes defective MUT is the deletion of an entire exon (coding region) corresponding to ∼120 base pairs (bp) (8). In this experiment, students use PCR to determine if a subject is affected by MMA due to exon deletion. At the beginning of the lab period, students, working in two-person teams, are provided the sequences of the native form of the MUT gene and the MUT gene with the ∼120 bp deletion. They are asked to design one set of PCR primers to distinguish the two forms of MUT and to determine PCR cycling conditions. These designs are subject to approval by the instructor. After students design their own primers, each student team is provided with a set of inexpensively purchased PCR primers, the native MUT gene, and an unknown MUT gene that may or may not be defective. (Plasmids encoding portions of the native and defective MUT genes are available gratis from this college or from Addgene for a nominal fee.) The students PCR amplify both samples and assess the PCR product size using agarose gel electrophoresis (9-12). If the same size fragment as the native MUT gene is observed, then the individual does not have MMA caused by exon deletion; a smaller fragment indicates the individual carries a mutated form of the MUT gene.
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Vol. 87 No. 10 October 2010 pubs.acs.org/jchemeduc r 2010 American Chemical Society and Division of Chemical Education, Inc. 10.1021/ed100243x Published on Web 07/28/2010
In the Laboratory Table 1. Primer Sets Designed by Students for PCR Amplification of MUT Genes Native PCR Producta Primer Set (A)
50 CTC ATT AAT GAA ATT G 30
Mutant PCR Productb
Expected (bp)
Observed (bp)
Expected (bp)
Observed (bp)
590
∼600
474
∼500
234
∼220
118
∼120
321
∼300
205
∼200
398
∼400
282
∼300
718
∼700
602
∼600
50 CTA TGG AAA AAG TCA 30 (B)
50 CTT TGG AAA TTA CCA G 30 50 AGG GAC AAT TTA CAT 30
(C)
50 GTC TCT AGA GTG GAT TAG 30 50 AGA GCC CAG TTC ACA G 30
(D)
50 GAA GAC AAG CTA GAA 30 50 AAA TCA AGC TAT TAA 30
(E)
50 GTA AAA CGA CGG CCA GT 30 50 CAG GAA ACA GCT ATG AC 30 c
a Native PCR product refers to PCR product of the functional MUT enzyme. b Mutant PCR product refers to the PCR product of the MUT enzyme containing an exon deletion. c Primer set E listed in the last row contains the sequences of commercially available M13 primers.
stressed that the data they collect could indicate the guilt or innocence of another person accused of murdering an infant by feeding the child poison. Additional details regarding the students' assignments, the procedures, and equipment used are provided in the supporting information. The experiment requires four laboratory hours for completion; 10, two-person student teams can be easily accommodated in one laboratory section with one thermocycler. Hazards
Figure 1. Representative image of an agarose gel used to analyze the PCR product sizes of the native MUT gene and the MUT gene with an exon deletion using different primer sets. Primer Set E is M13 primers that are commercially available. Cycling conditions were 25 cycles of: 95 °C for 20 s, 45 °C for 20 s, 72 °C for 45 s.
Sets of PCR primers designed by students to analyze the MUT gene and the corresponding expected and observed sizes of the PCR products are shown in Table 1. The last primer set in the table is for M13 primers that are commercially available. A representative image of an agarose gel used to analyze the PCR products is shown in Figure 1. Although there are several other published experiments for undergraduate biochemistry laboratories that use PCR (13-15), none is case-based or guides students through the process of experimental design. However, one of these studies does have a corresponding case that is used in the classroom (14). Another report describes an experiment for first-year medical students that uses PCR to determine if a patient has cystic fibrosis (16).
Students must wear safety glasses. They should also wear gloves when handling agarose gels that have been stained with ethidium bromide, a known carcinogen. This hazard can be avoided by using SYBR Safe DNA Stain (Invitrogen). It is especially important that safety glasses be worn when using a transilluminator or hand-held UV lamp to visualize the PCR products in the agarose gels. If a safety cabinet is not available, students should use face shields instead of safety glasses. Conclusion We have described a case-based laboratory for undergraduate biochemistry students. This experiment allows students to approach the laboratory in an exploratory manner, relying on their scientific skills and minimizing the step-by-step, “cookbook” approach. Literature Cited
Case Management The case study is used to introduce the experiment (available in the supporting information). It is provided to the students at least 2 days prior to their laboratory experience. They are told that the case they will work on parallels the Stevens' case. It is
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1. National Center for Case Study Teaching in Science. http://ublib. buffalo.edu/libraries/projects/cases/case.html (accessed Jul 2010). 2. Start With A Story; Herreid, C. F., Ed.; NSTA Press: Arlington, VA, 2007. 3. Dinan, F. J.; Szczepankiewicz, S. H.; Carnahan, M.; Colvin, M. T. J. Chem. Educ. 2007, 84, 617–618. 4. Hoffman, M. Science 1991, 254, 931. 5. Deodato, F.; Boenzi, S.; Santorelli, F. M.; Dionisi-Vici, C. Am. J. Med. Genet., Part C 2006, 142C, 104–112. 6. Mullis, K. B.; Faloona, F. A. Methods Enzymol. 1987, 155, 335–350. 7. PCR Primer: A Laboratory Manual; Dieffenbach, C. D., Dveksler, G. S., Eds.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 2003.
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8. Acquaviva, C.; Benoist, J. F.; Pereira, S.; Callebaut, I.; Koskas, T.; Porquet, D.; Elion, J. Hum. Mutat. 2005, 25, 167–176. 9. Aaij, C.; Borst, P. Biochim. Biophys. Acta 1972, 269, 192–200. 10. Brody, J. R.; Kern, S. E. Anal. Biochem. 2004, 333, 1–13. 11. Sambrook, J.; Russell, D. Molecular Biology: A Laboratory Manual, 3rd ed.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 2001. 12. Current Protocols in Molecular Biology; Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. A., Seidman, J. G., Smith, J. A., Struhl, K., Eds.; John Wiley and Sons, Inc.: New york, 2007. 13. Weller, D. L. J. Chem. Educ. 1994, 71, 340–341.
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14. Taylor, A.; Sajan, S. J. Chem. Educ. 2005, 82, 597–598. 15. Jackson, D. D.; Abbey, C. S.; Nugent, D. J. Chem. Educ. 2006, 83, 774–776. 16. Grody, W. W.; Kronquist, K. E.; Lee, E. U.; Edmond, J.; Rome, L. H. Am. J. Hum. Genet. 1993, 53, 1352–1355.
Supporting Information Available Case study; notes and procedures for instructors; additional details for cloning MUT genes used in the experiment; instructions for students. This material is available via the Internet at http://pubs.acs.org.
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