Karen M. Lounsbury From the Department of Pharmacology, University of Vermont, Burlington, Vermont 05405

2003 by The International Union of Biochemistry and Molecular Biology BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION Printed in U.S.A. Vol. 31, No. 1, pp. 37 41, 2003 Laboratory Exercises Crime Scene Investigation AN EXERCISE IN GENERATING AND ANALYZING DNA EVIDENCE* Received for publication, September 16, 2002, and in revised form, September 30, 2002 Karen M. Lounsbury From the Department of Pharmacology, University of Vermont, Burlington, Vermont 05405 The goal of this project is to introduce students to molecular biology techniques using an experimental setting that inspires both scientific and personal interest. The project is designed as a small group apprenticeship for gifted high school juniors or seniors who can spend full time in a sponsor s laboratory for at least 1 week. The students begin by examining evidence from a mock crime scene that consists of hair samples from the crime scene and from five potential suspects. Students extract DNA from the hair samples and amplify a hypervariable region within the mitochondrial genome using the polymerase chain reaction. Amplified products are then sequenced and compared with the crime scene sequence using DNA alignment software. In consecutive projects, students from four different schools successfully identified the suspect who matched the crime scene evidence. This project is a valuable learning tool not only due to the comprehensive introduction to molecular biology techniques but also because it helps the students to connect scientific exploration with well publicized media events and provides a window into potential career opportunities in the field of molecular biology. Keywords: DNA extraction, PCR, DNA evidence, forensic science. Through grants funded by the Howard Hughes Medical Institute (HELiX (Hughes Endeavor for Life Science Excellence), www.hhmi.org) and the National Science Foundation (Vermont EPSCoR (Experimental Program to Stimulate Competitive Research)), several researchers at the University of Vermont developed summer apprentice programs consisting of a 1 week intensive lab experience for two high school students and their science teacher. The goal was to train the students and teacher in techniques that they could then apply during the school year using equipment supplied by the HELiX program. At the end of the school year, the students competitively presented their results as posters and talks at the Science and Technology Career Day held for regional high school students at the University of Vermont. After agreeing to take part in this educational project, the first step was to choose an appropriate project that was 1) educational, 2) feasible, and 3) interesting to the students. Teaching molecular biology to secondary level students is not an easy task. DNA is an abstract idea akin to quantum physics, and its study can appear quite daunting to an inexperienced student. The project described here allowed students to learn DNA extraction, polymerase chain reaction (PCR) and sequencing using a mock crime scene to add interest and to relate the experience to both * This project was supported by Howard Hughes Medical Institute HELiX Program Grant HHMI522704 and National Science Foundation Vermont EPSCoR Program Grant EPS9874685. To whom correspondence should be addressed. Tel.: 802-656-1319; E-mail: klounsbu@zoo.uvm.edu. This paper is available on line at http://www.bambed.org 37 media events and potential career paths. Results of this experience demonstrated that students gained confidence in their ability to understand DNA, work with laboratory equipment, and analyze and present data. Perhaps more importantly, they also grew to appreciate the awesome power of molecular biology. The project uses a similar strategy to that used by forensic laboratories to distinguish DNAs from samples collected at a crime scene. For the experimental method, DNA was extracted from hair or cheek samples, and the hypervariable control region (HV1) within the mitochondrial gene, cytochrome b, was amplified and sequenced [1, 2]. Sequences were compared, and suspects were compared with the crime scene samples. EXPERIMENTAL PROCEDURES Introductory Materials Introduction to DNA Websites: www.dnaftb.org and www.dna. com. The FBI Forensic Website: www.fbi.gov/hq/lab/handbook/ examsdna.htm. Nobel Prize winners whose findings revolutionized molecular biology: Watson and Crick (DNA structure), Sanger (DNA sequencing), Nirenburg (genetic code), and Mullis (PCR). Also helpful if available: a tour of a local forensic laboratory and a teaching session at the DNA sequencing facility. A good reference text for molecular biology is An Introduction to Molecular Biology [3], and information on the molecular biology techniques can be found in Ref. 4, which also has on-line access. Introduction of the students to the laboratory setting included an orientation session where lab safety procedures, rules, and potential hazards were addressed. Because most students had never operated a Pipetman previously, all students were given a pipetting accuracy tutorial where various volumes of water were weighed on the balance using each size Pipetman. This training is

38 BAMBED, Vol. 31, No. 1, pp. 37 41, 2003 critical because the procedures below include several important small pipette measurements. Materials 1. Equipment: Pipetman (p20 and p200), sterile pipette tips, and microcentrifuge tubes (1.5 ml and PCR), waterbath (37 65 C), agarose gel electrophoresis set-up, microcentrifuge, PCR machine, UV lamp (for visualizing DNA in ethidium bromide gels), DNA sequencing facility access. 2. Reagents a. DNA extraction: Ice, 70% ethanol, isopropanol, 20 mg/ml Proteinase K (Promega, Madison, WI), 0.5 M EDTA (Sigma), Wizard DNA purification kit (Promega), Pellet Paint TM (optional but useful; Novagen, Madison, WI). b. PCR: PCR kit (Promega), dntp mix (Promega), individual nucleotides mixed to make 10 mm stock solution, PCR primers (Operon, Almeda, CA) made to 10 M stock solution: Forward F15971: 5 -TTAACTCCACCATTAGCACC Reverse R16410: 5 -GAGGATGGTGGTCAAGGGAC c. Electrophoresis: 10 DNA Sample Buffer (Brinkmann, Westbury, NY), 100-bp DNA ladder (Promega), electrophoresis grade agarose (Sigma), 10 mg/ml ethidium bromide (Sigma), TAE buffer (40 mm Tris-HCl, 0.1% glacial acetic acid, 1 mm EDTA), Qiaquick Gel purification kit (Qiagen, Alameda, CA). d. Sequencing: Same primers as PCR, no additional reagents. Method Notes for Students Wear latex or preferably neoprene gloves for all procedures to avoid contamination of samples with DNA from your fingers and to avoid exposing your skin to potentially toxic substances. Use heat protection when handling hot agarose. Always wear UV protection for eyes when visualizing DNA in gels. Potential Hazards Ethidium bromide is a suspected carcinogen and should be handled carefully with gloves. Check local procedures for disposal. UV light exposure can cause burns to skin around goggles; wear full-face protection if possible or limit exposure. a. DNA extraction and purification (adapted from Promega s Wizard DNA Purification for Tissue) 1. Obtain at least 15 hairs from volunteer suspects (30 from designated criminal). It is useful to choose volunteers from diverse ethnic backgrounds to increase odds of differences in sequence. Trim the hair close to the head. Colored hair is not a problem, but do not use hair from the shower drain as soap residue interferes with DNA extraction. 2. Rinse in 70% EtOH for 30 min followed by an H 2 O rinse for 30 min (removes hair treatments). 3. For each sample to be processed, add 120 l of0.5m EDTA (ph 8.0) to 500 l of Nuclei Lysis solution (from Wizard kit). 4. Cut hair into small pieces (we used a glass plate and razor blade) and transfer to a sterile microcentrifuge tube containing 600 l of EDTA/Nuclei Lysis solution. 5. Add 17.5 l of 20 mg/ml Proteinase K and incubate overnight (or longer) at 56 C (this breaks disulfide bonds and digests protein). The sample solution should appear cloudy after incubation. The laboratory sponsor should prepare the samples prior to arrival of students and then have them repeat the procedure to generate data in duplicate. 6. Add 3 l of RNase solution (from Wizard kit) to the nuclear lysate and mix the sample by inverting the tube five times. Incubate the mixture 15 30 min at 37 C. Allow the sample to cool to room temperature for 5 min before proceeding. 7. To the room temperature sample, add 200 l of Protein Precipitation solution (from Wizard kit) and vortex vigorously at high speed for 20 s. Chill the sample on ice for 5 min and then centrifuge for 4 min at 13,000 g. The precipitated protein will form a white pellet. 8. Carefully remove the supernatant containing the DNA (leaving the protein pellet behind) and transfer it to a clean 1.5-ml microcentrifuge tube containing 600 l of room temperature isopropanol. Add 2 l of Pellet Paint to supernatant to more easily visualize the DNA pellet. 9. Gently mix the solution by inversion and then centrifuge for 1 min at 13,000 g at room temperature. The DNA will be visible as a small pellet (pink if using Pellet Paint). Carefully decant and discard the supernatant. 10. Carefully add (to avoid dislodging pellet) 600 l of room temperature 70% ethanol. Centrifuge for 1 min at 13,000 g at room temperature. 11. Aspirate the ethanol using a Pipetman. The DNA pellet is very loose at this point and care must be used to avoid aspirating the pellet. 12. Invert the tube on a Kimwipe (Kimberly-Clark Corp.) and air-dry the pellet for 10 15 min. 13. Add 15 l of DNA Rehydration solution (from Wizard kit) and incubate at 65 C for 1 h. Periodically mix the solution by gently tapping the tube. This solution is the Template DNA. Store the Template DNA at 4 C (or freeze if storing for more than a couple of days). b. PCR 1. Mix the following in a dome-capped PCR tube: 33 l of sterile H 2 O 5 l of10 Taq Buffer 1 l of10mm dntp mix 2.5 l of10 M Forward Primer 2.5 l of10 M Reverse Primer 5 l of Template DNA (your sample) 1 l oftaq DNA Polymerase 2. PCR conditions: Denature 94 C 30 s, cycle (94 C 20 s, 56 C 10s,72 C 30s) 32, final extension 72 C 2 min, soak 4 C. c. Agarose gel electrophoresis 1. Prepare the gel a) Weigh out 0.6 g of agarose powder into a 250-ml flask. b) Add 50 ml of 1 TAE buffer and loosely plug flask with paper towel or something similar. c) Microwave 30 s at 80%, swirl flask, repeat another 30 s until completely dissolved. NOTE: agarose can easily become superheated in the microwave so use caution when swirling the flask and wear a heat-protective glove. Agarose can alternatively be dissolved using a hot plate with a stir bar. d) Add 5 l of 10 mg/ml ethidium bromide (see Potential Hazards above). Let cool until the flask is hot but not burning ( 55 C). Note: pouring the gel when it is too hot may warp the electrophoresis mold, but pouring it too cool will cause an uneven gel matrix. e) Pour into gel mold. Place comb in gel. Let gel about 30 min. Remove comb carefully, put the gel into the gel box (wells toward the black ( ) electrode), and fill with 1 TAE buffer until the gel is covered.

39 FIG. 1.Loading the agarose gel. A Pipetman is used to carefully load the PCR products into the gel wells. The DNA Sample Buffer contains glycerol to make the sample dense, so it sinks into the well instead of diffusing. The bromphenol blue dye tracks the gel front, so the separation distance can be monitored. 2. Prepare samples a) Make a stock solution of 100-bp DNA ladder by mixing 10 l of 100-bp DNA ladder, 10 l of10 DNA Sample Buffer, and 80 l of distilled water. b) Add 5.5 l of10 DNA Sample Buffer to each PCR sample and tap the tube to mix. 3. Load and run the gel a) Use a Pipetman to load 10 l of DNA marker into the first well (Fig. 1). Load 40 l of the PCR samples into the adjacent wells. If there are enough wells, skip a well between each sample to reduce the chance of contamination between samples. b) Check the orientation of the gel to ensure that the wells are at the black ( ) end and the current will move toward the red ( ) end. Note: DNA is negatively charged, so it will move toward the positive electrode under current. c) Place the cover over the apparatus, and again check the electrode orientation: run to red. Plug in the electrodes and apply voltage: 100 V for 30 min, 50 V for 1 2 h, and 10 V for overnight. d. Gel extraction and purification (adapted from Qiaquick Gel purification kit (Qiagen), kit contains Buffers GQ, PE, and EB) 1. Remove the gel from the electrophoresis apparatus and place onto the UV light box. Visualize fluorescent DNA bands (wear eye protection). The DNA marker should have several bands that represent 100 700-bp DNA (smaller DNA runs faster). The PCR sample lanes should have a single band at 430 bp and a light smudge below 100 bp (PCR primers). 2. Using a fresh razor blade for each sample, cut out the 430-bp band with as little excess gel as possible. Limit exposure to UV light as much as possible. 3. Transfer the gel piece to a fresh 1.5-ml microcentrifuge tube and weigh on a balance that has been tared with the empty tube. Weight between 0.1 and 0.3 g is optimal. 4. Add 3 volumes of Buffer GQ to the tube (i.e. 0.1 g 0.1 ml, so 3 volumes 0.3 ml or 300 l). 5. Incubate at 50 C for 10 min (vortex every 2 3 min). 6. Check color (yellow is good, see handbook if not) and make sure gel piece is completely melted. 7. Add 1 volume of isopropanol and mix. 8. Place Qiaquick spin column in collection tube and label the top. 9. Add sample and centrifuge 13,000 g for 1 min. If the sample has too much volume, centrifuge half and then load the rest and centrifuge again. 10. Discard the flow-through. 11. Add 500 l of Buffer GQ and centrifuge for 1 min. 12. Add 750 l of Buffer PE, let stand 2 5 min, and centrifuge for 1 min. 13. Discard the flow-through and centrifuge again for 1 min. 14. Place column into a clean 1.5-ml microcentrifuge tube and label. 15. To elute the DNA, add 30 l of water (not Buffer EB) to the center of the paper disk at the bottom of the column. Let stand 1 min and then centrifuge for 1 min. Store at 20 C. Note: using water at this step is better for automated sequencing. e. Sequencing and analysis 1. Send the DNA to the sequencing analysis facility (many facilities are available at universities throughout the country). Optimally, send on either dry or wet ice, but samples can survive room temperature for a couple of days. Also supply an aliquot of the Forward PCR primer (25 l). Inform the facility that your samples are PCR products. 2. Use DNA analysis software such as Sequencher TM in the DNA facility to align suspect sequences to samples found at the crime scene. Note: see comments on heteroplasmy under Results. RESULTS Successful Identification of Suspect Linked to Crime Scene Three independent groups of students and teachers were successful in generating PCR products from 80% of the hair samples. The samples that were most difficult were very coarse and did not digest well in the first step. In one unsuccessful case, the hair sample was obtained from the shower drain, suggesting that soaps and detergents may interfere with the extraction. A follow-up on these samples using cheek swabs was successful in all cases. The use of Pellet Paint was extremely helpful in the DNA extraction because the tiny DNA pellets were easily visualized for instant gratification. There were surprisingly few occasions where a DNA pellet was not obtained even when using small amounts of starting material. In all but one sample, if a DNA pellet was visible, a PCR product was obtained. PCR products were easily visualized following agarose electrophoresis (Fig. 2). The students tended to take longer than expected to extract the gel pieces, which caused excess exposure of unprotected skin to the UV light. For this reason, a full face shield is recommended. DNA sequences were obtained for all of the samples tested using the forward primer from the PCR step. An alignment of the sequences showed few base changes; however, there were multiple differences in heteroplasmic bases (the occurrence of two co-dominating sequences in the same individual), denoted as non-calls on the sequence readout (Fig. 3). Maternally related individuals shared the same heteroplasmic positions, suggesting that these positions can be used to determine differences between sequences. Common heteroplasmy has been dem-

40 BAMBED, Vol. 31, No. 1, pp. 37 41, 2003 school year, and they received class credit for the work. This group was successful in generating a tremendous amount of data, and their efforts were rewarded when they received the top award for their presentation at Science and Technology Career Day at the University of Vermont. FIG. 2.Extracting PCR product from the gel. Ethidium bromide in the gel binds to DNA, allowing visualization of the PCR product as a UV fluorescent band. Bands are cut with a razor blade and placed into a microcentrifuge tube for purification. FIG. 3. Sample DNA sequence of PCR for mtdna in HV1 region. The DNA chromatogram gives the sample sequence. Sequences are then analyzed using DNA comparison software. An arrow denotes an example of base heteroplasmy that was used to distinguish differences between samples. onstrated by others for this region of the cytochrome b gene [5, 6]. In the three separate crime scene investigations, using different suspects, the heteroplasmic differences were successfully used to correctly identify the source of hair sample found at the crime scene. Problems and Solutions The most difficult portion of this project was the continuation of the project at the home high school laboratory. Without an experienced mentor, several problems arose that took a frustratingly long time to resolve. Examples of problems included: incorrect buffer dilutions, malfunctioning equipment, improper handling of DNA, improper storage of reagents, inability to quantify PCR products, and loss of samples in transit to the sequence facility. Although these problems were all overcome successfully, a visit to the high school laboratory to help get things set up is highly recommended to circumvent many of these setbacks. Another problem was the lack of significant time in the students busy schedules to complete the work. Some groups attempted intensive labs during school holidays; however, troubleshooting in these cases was nearly impossible due to lack of time. The most successful group set aside 3 h/week to work on the project for the entire DISCUSSION The crime scene investigation laboratory exercise was very successful from the standpoint of interest, collection of data, interpretation of data, and hands-on techniques. The concepts explored included DNA extraction from small amounts of starting material, PCR to amplify small quantities of DNA, and sequencing to establish differences among individuals. Presentations by the students demonstrated that they had increased their knowledge of molecular biology techniques and career applications. Sponsor Observations It was very easy to get the students interested in the use of molecular biology techniques to solve a crime. The simplicity of the goal to link crime scene data to a limited number of suspects prevented the students from becoming overwhelmed. Students quickly adjusted to the new instrumentation, and pipette practice by weighing water was time well spent. The biggest accomplishments of the laboratory exercise were the technical successes and the connection between molecular biology and familiar media stories. Students remarked on the possibility of the data accumulated in a real crime situation and were impressed by the responsibility of the scientists performing the DNA analysis. The least successful area was teaching concepts related to solution component calculations and their roles in the procedure. Students and teachers alike stumbled in situations where new buffers needed to be prepared or diluted. These situations also contributed to problems when trying to reproduce experiments at the home school. These observations revealed that the area of weakness in mathematical calculations needs to be heavily instructed in undergraduate and graduate laboratory experiences but that time is better spent at the secondary education level with technical aspects and concepts. Student Feedback A questionnaire was distributed to the students following the completion of the project. Students and teachers alike were impressed with the use of high tech facilities at the University of Vermont as compared with their limited laboratory resources. The students especially enjoyed the environment of staying in the University dorms and enjoying the chaperoned college experience during their project. The hands-on aspect was also highly praised as was the organization of the project to ensure that data were obtained. Similar to the problems discussed above, the primary criticism of the project was the lack of guidance at their home school. Overall the project was highly successful at stimulating student interest in molecular biology research. The experimental set-up was not difficult for any laboratory with standard equipment for performing molecular biology techniques. Obtaining enough starting material was not an issue, and sequences were easily distinguished in the hypervariable region examined. From a mentoring standpoint, it was very rewarding to have the opportunity to

41 share interests and technical expertise with students that have not yet set their future course. The overall success of this project is an example that encourages expansion of funding for these types of programs to increase the number of students that can participate in a small apprenticeship program. Acknowledgments DNA sequencing was performed by the Vermont Cancer Center DNA Facility at the University of Vermont. I thank Gayle Bress, University of Vermont, for organizing the program and the energetic high school participants from Westport Central School, New York, Rutland High School, Vermont, Mill River Union High School, Vermont, and Poultney High School, Vermont. I also thank the Vermont State Forensic Laboratory and Timothy Hunter of the Vermont Cancer Center DNA Facility for informative teaching sessions. REFERENCES [1] K. W. Miller, J. L. Dawson, E. Hagelberg (1996) A concordance of nucleotide substitutions in the first and second hypervariable segments of the human mtdna control region, Int. J. Legal Med. 109, 107 113. [2] R. J. Steighner, M. Holland (1998) Amplification and sequencing of mitochondrial DNA in forensic casework, Methods Mol. Biol. 98, 213 223. [3] R. Tait (1997) An Introduction to Molecular Biology, Horizon Scientific Press, Wymondham, Norfolk, UK. [4] J. Sambrook, D. Rusell (2001) Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. [5] A. Salas, M. V. Lareu, A. Carracedo (2001) Heteroplasmy in mtdna and the weight of evidence in forensic mtdna analysis: a case report, Int. J. Legal Med. 114, 186 190. [6] T. Grzybowski (2000) Extremely high levels of human mitochondrial DNA heteroplasmy in single hair roots, Electrophoresis 21, 548 553.