Solving a Crime Using DNA Analysis and Chemistry (basic forensics): Instructor s Manual

Transcription

1 Solving a Crime Using DNA Analysis and Chemistry (basic forensics): Instructor s Manual I. Purpose and Concepts Covered...1 II. Protocol for Crime Scene Investigation Using RFLP Analysis...2 A. Preparation for the Laboratory...2 B. Teaching Suggestions...3 C. The Scenario: The Last Bag of Chocolate Chips...4 D. Chromatography of Marker Inks...5 E. RFLP Analysis of DNA Samples...6 III. STR-Based Analysis of DNA Using Silver-Stained Gel...9 A. Before You Begin...9 B. Amplification...10 C. Polyacrylamide Gel Preparation...12 D. Polyacrylamide Gel Electrophoresis...15 E. Silver Staining...17 F. Generating Film Images...19 G. Analyzing Data...20 H. Obtaining Single-Source Human Genomic DNA...23 IV. STR Analysis of DNA Using the PowerPlex 16 System...24 V. Supplier and Ordering Information...25 VI. Resources...26 This instructor s! manual is avaliable online only. This teaching resource is made available free of charge by Promega Corporation. Reproduction permitted for noncommerical educational purposes only. Copyright 2009, 2010 Promega Corporation. All rights reserved. I. Purpose and Concepts Covered This introductory forensics laboratory is for use in courses that cover basic topics in molecular biology and genetics. While modern DNA-based forensics uses short tandem repeat (STR) analysis, the first lab in this manual uses restriction fragment length polymorphism (RFLP) analysis. RFLP analysis is the conceptual predecessor for modern STR analyses conducted by crime scene investigators and medical laboratories. We have included simple chromatography analyses of evidence in addition to the DNA analysis to remind students that crime scene evidence includes more than DNA. This teaching unit has a second laboratory for instructors of more advanced students. The second laboratory is an STR-based laboratory and will require access to sophisticated laboratory equipment including a polyacrylamide gel apparatus. Instructors may choose to run all or part of this lab as a demonstration for students. Page 1

2 II. Protocol II.A. Preparation for the Laboratory Note to the Instructor The identification of the "perpetrator" of the crime is based on RFLP analysis, a concept that is similar to STR analysis, which is used to identify individuals in modern forensics laboratories. A completely separate laboratory protocol for institutions that have the ability to conduct STR-based analyses is also included in this unit. The main exercise in this laboratory is the RFLP analysis of DNA. However, additional evidence may be analyzed, including the note found at the crime scene. The simple chromatography experiment is included to encourage students to view DNA evidence as one piece of the puzzle of a crime scene. You can work with a chemistry faculty member to "dress up" the chemical analysis of the crime scene, if you would like to cover more complex chemical topics along with the DNA analysis. Materials Required For Chromatography Experiments crime scene tape empty glass (one for each team of students) two black markers of different brands (one each for each team of students) 500 ml beakers (two for each team of students) acetone gloves 3 mm Whatman filter paper hole punch glass stiring rod For RFLP Analysis XmnI restriction enzyme (5 units or 0.5 µl at 12 u/µl concentration) HincII restriction enzyme (6 units or 1.5 µl at 10 u/µl concentration) nuclease-free water Buffer B (restriction enzyme buffer) DNA sample from crime scene (pgl4.11[luc2p] Vector, Cat.# E6661; 1 µg for each team of students) DNA sample from suspect one (pgl4.11[luc2p] Vector, Cat.# E6661; 1 µg for each team of students) DNA sample from suspect two (pgl4.12[luc2cp] Vector, Cat.# E6671; 1 µg for each team of students) Agarose LE, Analytical Grade (Cat.# V3121) TBE Buffer, 10X (Cat.# V4251) ethidium bromide stock solution, 10 mg/ml DNA gel electrophoresis apparatus and power supply UV light box and camera or scanner pipettors and pipet tips 1.5 ml sterile tubes DNA markers (BenchTop pgem DNA Markers Cat.# G7521) gloves acetylated BSA (provided with restirction enzymes) 37 C water bath 65 C water bath Blue/Orange Loading Dye, 6X (Cat.# G1881) Page 2

3 II.B. Teaching Suggestions 1. Personalize this scenario with names and locations from your institution, or if you have another idea for a crime scenario that will work with this laboratory, write your own case to solve. 2. Create a learning community around this laboratory. Have genetics and chemistry students perform the scientific analysis. Ask students in prelaw, criminal justice or other appropriate majors to "prosecute" the case. The case can go to trial before a jury of peers from the college campus. Science students can act as "expert" witnesses to explain the evidence and testing to the rest of the community. Mass communications students can "cover" the case. 3. This laboratory builds on several other laboratories. For instance, DNA has to be isolated from a crime scene before it can be analyzed. Consider leading your students through Unit 4: Genomic DNA Purification before performing this laboratory to give them background in DNA isolation. Additionally, STR analysis requires amplification of DNA using PCR. PCR is introduced in Unit 2: The Chemistry of Inheritance. These units are available at Each of these other units has laboratories, lectures and other teaching materials like animations that can be used in concert with this laboratory. 4. Have the students make all of their stock and working solutions to reinforce moles-to-gram concepts taught in basic chemistry. Page 3

4 II.C. The Scenario: The Last Bag of Chocolate Chips The manager of the college food services promised the president of the college a plate of his favorite chocolate chip cookies for the upcoming meeting with the executive council of the Board of Directors. The chocolate chip cookies are a specialty of Chef Lombardo's and are famous across the college campus. Almost like Pavlov's dogs, students, professors, faculty and staff emerge from their offices and dorm rooms in a grand migration toward the Dining Hall when the aroma of these cookies baking drifts across campus. The appeal of these cookies can be traced, in part, to the chocolate chips that are imported from Europe. The day before the board meeting, Chef Lombardo checked the pantries in the main kitchen and discovered that he only had one bag of these special chocolate chips left. He placed all of the ingredients onto the lower shelf of the pantry so that he would be ready to bake the cookies first thing the next morning. Chef Lombardo locked the pantry, turned off the kitchen lights and left for the night. When Chef Lombardo returned the next morning, he was greeted with a horrific sight. The door to the kitchen had been pried open, and the metal door on the pantry was bent and torn where someone had pried the lock of the pantry door. The ingredients for the cookies were scattered around the kitchen. The bags of sugar and flour were busted on the floor. The open and empty bag of chocolate chips lay on the kitchen counter along with a glass that had lipstick marks on it and a note printed in black marker that read: This is just to say I have eaten the chocolate that was in the pantry and which you were probably saving for tomorrow. Forgive me it was delicious so sweet and so warm. Apparently the thief had a literary bent, having adapted text from William Carlos Williams for the thank you note. The thief had cut his or her hand on the pantry door and left blood stains. Chef Lombardo immediately called the college president to report the tragedy. The criminal justice, genetics and general chemistry professors were all then called to the crime scene. The investigators interviewed a group of students who had returned home late from an outing and said that they had seen two professors walking back to campus at 2:00 am. The professors were walking toward the dining hall. One of the professors, Dr. Johnson in history, sat on the executive council as the faculty representative to the board and would have known about the chocolate chip cookies for the meeting. Page 4

5 Interestingly, both Dr. Johnson and the other professor, Dr. Lundquist in English Language Arts, each had a hand bandaged when they were interviewed. The chemistry professor confiscated the black markers that were on each professor's desk for further analysis. Each professor also gave buccal swab sample for DNA analysis. II.D. Chromatography of Marker Inks Before the Lab 1. Create a mock crime scene with evidence for your students to analyze. On a piece of 3 mm Whatman filter paper use one of the markers to write the thank you note found at the crime scene. Be sure to leave enough room between the bottom of the filter paper and the writing (about 2.5 cm) for chromatography. Make sure that the writing covers a sufficient area that the note can be cut into strips and distributed to each team of students. 2. Each team of students should receive two markers, representative of the kind collected from the suspects. Lab Protocol Note: Be sure to wear gloves when you handle the thank you note and your own filter paper. 1. Obtain a cut strip from the thank you note found at the crime scene. 2. Cut a clean piece of filter paper into two strips. Draw a line across the width of the first strip using one of your sample markers. Do the same thing with the other strip and the other marker. Your mark should be about 2.5 cm above the bottom of the strip. 3. Make a hole using a hole punch at the top of your evidence and sample strips. 4. Space all three strips along a glass stirring rod. 5. Carefully add acetone to the beaker so that it will just cover the bottom 0.5cm of the filter paper strips. The exact volume will depend on how far down the strips extend into the beaker when they are suspended by the glass stirring rod. 6. Allow the acetone to wick up the filter paper strips. Once the acetone has reached the top of the strips, remove them from the beaker and allow them to dry. (Place them on a nonabsorbent surface or hang them to dry). Analysis 1. Can you see a difference between the inks of the two sample markers? 2. Does either one of them clearly give the same chromatography pattern as the ink on the note? 3. Can you determine which type of marker was used to write the note? Why or why not? If you think additional experiments are needed, what would you do? Page 5

6 II.E. RFLP Analysis of DNA Samples Before the Lab 1. Distribute the crime scene DNA, suspect 1 DNA and suspect 2 DNA to the students. 2. Label 9 tubes for the following restriction enzyme digests: Reaction Tube # Crime scene sample Hinc II digest 1 Crime scene sample Xmn I digest 2 Crime scene sample HincII/XmnI digest 3 Suspect 1 sample Hinc II digest 4 Suspect 1 sample XmnI digest 5 Suspect 1 sample Hinc II/XmnI digest 6 Suspect 2 sample Hinc II digest 7 Suspect 2 sample XmnI digest 8 Suspect 2 sample Hinc II/XmnI digest 9 Restriction Enzyme Digestion of DNA Samples 1. Prepare the following master mixes for your single-enzyme digests as directed in the table below. Prepare enough master mix for 8 digests to compensate for pipetting errors. Master Mix for Single Digests Volume Needed Component for Each Reaction Volume Needed for 8 Reactions Nuclease-free water 16.3 µl µl 10X Buffer B 2.0 µl 16.0 µl Acetylated BSA 0.2 µl 1.6 µl 2. To the three HincII digests (Tubes 1, 4 and 7), add the following: Single-Digest Master Mix 18.5 µl DNA (1µg/ml) 1.0 µl Hinc II (12 U/µl) 0.5 µl 3. To the three XmnI digests (Tubes 2, 5 and 8), add the following: Single-Digest Master Mix 18.5 µl DNA (1 µg/ml) 1.0 µl Xmn I (10 U/µl) 0.5 µl 4. Prepare the master mix for your double-enzyme digests according to the table below. Prepare enough Master Mix for 5 digests to compensate for pipetting errors. Master Mix for Double Digests Component Volume Needed for Each Reaction Volume Needed for 5 Reactions Nuclease-free water 15.8 µl 79.0 µl 10X Buffer B 2.0 µl 10.0 µl Acetylated BSA 0.2 µl 1.0 µl Page 6

7 5. To the three double-enzyme digests (Tubes 3, 6 and 9), add the following: Double-Digest Master Mix 18.0 µl DNA (1µg/ml) 1.0 µl Hinc II (12 u/µl) 0.5 µl Xmn I (10 u/µl) 0.5 µl 6. Incubate the digests for 2 hours at 37 C. 7. Heat inactivate the reactions by placing the tubes at 65 C for 15 minutes. Notes: The double digest will result in 5 fragments for pgl4.12 and 4 fragments for pgl4.11. This is because Xmn I does not cut pgl4.11. Fragments from double digest of pgl4.12: 2215bp, 1306bp, 463bp, 328bp, and 110bp. Fragments from double digest of pgl4.11: 1306bp, 740bp, 110bp and 2214bp. Agarose Gel Electrophoresis of Restricted DNA 1. Prepare 1X TBE for your Gel Running Buffer. 2. Weigh out the required amount of agarose, and add it to the appropriate amount of 1X TBE buffer in a flask or bottle. For example to prepare a 2% agarose gel, add 2.0 g of agarose to 100 ml of buffer. 3. Heat the mixture in a microwave oven or on a hot plate for the minimum time required to allow all of the agarose to dissolve. Interrupt the heating at regular intervals, and swirl the contents. Do not allow the solution to boil over. 3. Cool the solution to C and pour the gel. Be sure to insert a gel comb to create sample wells. Allow the gel to cool completely. Remove the comb from the gel and place the gel in the electrophoresis apparatus. 4. To analyze samples on the gel, prepare the following: Enzyme digest 5 µl Blue/Orange Loading Dye, 6X 1 µl 5. Add enough 1XTBE gel running buffer to cover the gel. 6. Load the samples onto the gel, and run at the voltage recommended by the the gel box manufacturer. 7. Run the gel until the orange dye front (runs at approximately the same rate as a 50 bp piece of DNA) is near the bottom of the gel. 8. Remove the gel, and stain it by soaking it in a solution of 0.5 µg/ml ethidium bromide (this is diluted from the 10 mg/ml) for 30 minutes at room temperature. Note: Ethidium bromide is a carcinogen. Wear gloves and, be sure to dispose of it in accordance with your institution s guidelines. 9. Place the gel on a UV light box, and photograph the gel. Wear protective eyewear when using the UV light box. If the gel is too orange you can destain the gel in water for a few minutes at room temperature. Page 7

8 Analysis 1. Do either of your suspect DNA samples have the same RFLP pattern as the crime scene sample? 2. The two DNA samples probably have different restriction fragments. Can you figure out why (what is the polymorphism that you were able to detect)? 3. What is the purpose of the heat inactivation step at the end of the reaction? 4. How does agarose gel electrophoresis separate DNA fragments? 5. TBE buffer is composed of tris, borate and EDTA. What function does each of these ingredients serve in the buffer? 6. What is the function of the running buffer during agarose gel electrophoresis? Can you use deionized water instead? 7. Many chemicals stain DNA. Compare the way the following DNA stains work: ethidium bromide, propidium iodide, methylene blue and DAPI. Marker Suspect 1 HIncII Suspect 1 Double Suspect 1 XmnI Suspect 2 HIncII Suspect 2 Double Suspect 2 XmnI Scene HincII Scene Double Scene XmnI RFLP analysis of crime scene and suspect DNA. Page 8

9 III. STR-Based Analysis of DNA and Silver Stain Gel III.A. Before You Begin This protocol will require running a polyacrylamide gel. You will need glass plates and the accompanying gel electrophoresis apparatus. You can purchase precast 6% polyacrylamide gels from GE Life Sciences and Invitrogen, but you will also need the appropriate gel electrophoresis apparatus for the precast gels. The quality of the purified DNA sample and choice of thermal cycler, as well as small changes in buffers, ionic strength, primer concentrations, and thermal cycling conditions, can affect amplification. We suggest strict adherence to recommended procedures for amplification, denaturing gel electrophoresis, silver stain analysis and recording data on film. PCR-based STR analysis is subject to contamination by very small amounts of human DNA. Extreme care should be taken to avoid cross-contamination when preparing sample DNA, handling primer pairs, setting up amplification reactions and analyzing amplification products. Reagents and materials used prior to amplification (STR 10X Buffer, K562 Control DNA and 10X Primer Pairs) are provided in a separate box and should be stored separately from those used following amplification (allelic ladders, STR 2X Loading Solution and pgem DNA Markers). Always include a negative control reaction (i.e., no template) to detect reagent contamination. We highly recommend the use of gloves and aerosol-resistant pipette tips. Some of the reagents used in the analysis of STR products are potentially hazardous and should be handled accordingly. Table 1 describes the potential hazards associated with such reagents. Table 1. Hazardous Reagents Reagent acetic acid (fix/stop solution) acrylamide ammonium persulfate bisacrylamide formaldehyde (staining solution and developer solution) formamide (STR 2X Loading Solution) methacryloxypropyltrimethoxysilane (bind silane) silver nitrate (staining solution) sodium thiosulfate (developer solution) TEMED urea xylene cyanol FF (STR 2X Loading Solution) Hazard acetic acid (fix/stop solution) suspected carcinogen, neurotoxin oxidizer, corrosive toxic, irritant highly toxic, suspected carcinogen irritant, teratogen toxic, moisture sensitive highly toxic, oxidizer irritant, hygroscopic corrosive, flammable irritant irritant Note: Be sure to follow your institution s safety guidelines and procedures for using and disposing of hazardous materials. Note: To avoid working with unpolymerized acrylamide and several other chemicals in Table 1, use precast acrylamide gels. Remember that you will need a gel electrophoresis apparatus that can accomodate whatever precast gel you chose. Page 9

10 III.B. Amplification Note: This STR-based protocol will not work with the pgl4 plasmids used in the RFLP laboratory (Section II). You must use singlesource human genomic DNA for this laboratory. See Section III.H for suggestions for DNA sources. The GenePrint STR Systems were developed for amplification without artifacts usingtaq DNA polymerase. Use the buffer provided in the kit for your amplifications with Taq DNA polymerase. This protocol is for the amplification of CTT and amelogenin, allowing your students to profile three STR loci and determine whether the DNA is of a male or female origin. Materials to Be Supplied by the User GenePrint System (CSF1PO, TPOX, TH01; Cat.# DC6001 GenePrint Sex Identification, Amelogenin (Silver Detection; Cat.# DC4081) bind silane silver nitrate formaldehyde, 37% sodium thiosulfate, 10mg/ml sodium carbonate thermal cycler, model 480 or GeneAmp system 9600 (Perkin-Elmer) microcentrifuge Taq DNA polymerase (GoTaq DNA Polymerase Cat.# M3001) Nuclease-Free Water (Cat.# P1193 or equivalent) Mineral Oil (Cat.# DY1151 or equivalent) 0.5 ml or 0.2 ml microcentrifuge tubes (compatible with thermal cycler) 1.5 ml microcentrifuge tubes BSA Fraction V (optional) aerosol-resistant pipette tips crushed ice The CTT multiplex and GenePrint Sex Identification System, Amelogenin are optimized for use with GeneAmp reaction tubes and the Perkin-Elmer model 480 thermal cycler. When using a thermal cycler on which a system was not optimized, there may be a loss in product yield or sensitivity, and the balance between loci may change slightly. Meticulous care must be taken to ensure successful amplification. See our Web site or contact Technical Services for help optimizing amplification conditions. Amplification Setup We highly recommend that you wear gloves and use aerosol-resistant pipet tips to prevent contamination. 1. Thaw the STR 10X Buffer and 10X Primer Pairs, and place on ice. Note: Mix reagents by vortexing for 15 seconds before each use. 2. Place one clean, autoclaved 0.5 ml reaction tube for each reaction into a rack, and label appropriately. 3. Determine the number of reactions to be set up. This should include a positive and negative control reaction. Add 1 or 2 reactions to this number to compensate for pipetting error. While this approach does consume a small amount of each reagent, it ensures that you will have enough PCR maste mix for all samples. 4. Calculate the required amount of each component of the PCR master mix (Table 2). Multiply the volume (µl) per sample by the total number of reactions (from Step 3) to obtain the final volume (µl). Page 10

11 III.B. Amplification (continued) 5. In the order listed in Table 2, add the final volume of each reagent to a sterile tube. Mix gently (do not vortex), and place on ice. Table 2. Combined CTTv Multiplex and Amelogenin Reactions PCR Master Mix Component Volume Per Sample (µl) sterile water STR 10X Buffer 2.50 CTT Multiplex 10X Primer Pair 2.50 Amelogenin 10X Primer Pair 2.50 Taq DNA Polymerase (5u/µl) 0.15 (0.75 u) Total volume = Number of Reactions Final Volume (µl) Note: The volume given assumes a Taq DNA polymerase concentration of 5 u/µl. For different enzyme concentrations, the volume of enzyme addedmust be adjusted accordingly. If the final volume of Taq DNA polymerase added to the master mix is less than 0.5 µl, you may wish to dilute the enzyme with STR 1X Buffer, and add a larger volume. The amount of sterile water should be adjusted accordingly so that the final volume per reaction is 25 µl. Do not store diluted Taq DNA polymerase. 6. Add 22.5 µl of PCR master mix to each tube, and place on ice. Failure to keep the reagents and samples on ice can produce imbalanced amplification of multiplexed loci. 7. Pipet 2.5 µl of each sample into the respective tube containing 22.5 µl of PCR master mix. 8. For the positive amplification control, pipet 2.5 µl (5ng) of K562 DNA (diluted to 2 ng/µl) into a 0.5ml reaction tube containing 22.5 µl of PCR master mix. 9. For a negative amplification control, pipet 2.5 µl of sterile water (instead of template DNA) into a 0.5ml reaction tube containing 22.5 µl of PCR master mix. 10. If you are using a thermal cycler with an unheated lid, add 1 drop of mineral oil to each tube. Close the tubes. Note: Allow the mineral oil to flow down the side of the tube and form an overlay to limit sample loss or cross-contamination due to splattering. 11. Centrifuge the samples briefly to bring the contents to the bottom of the tube. Page 11

12 Thermal Cycling Protocol 1. Place the tubes in a thermal cycler. 2. Run the protocol below: Initial Incubation: 96 C for 2 minutes Programmed Ramp Times: None First 10 Cycles 94 C for 1 minute 64 C for 1 minute 70 C for 1.5 minutes Programed Ramp Times: None Last 20 Cycles 90 C for 1 minute 64 C for 1 minute 70 C for 1.5 minutes Extension Step: None Hold at 4 C. 3. After completing the thermal cycling protocol, store the samples at 20 C. Note: Storing the amplification products at our above 4 C may result in degradation products. III.C. Polyacrylamide Gel Preparation Materials to Be Supplied by the User 40% acrylamide:bis (19:1) and TEMED TBE Buffer, 10X (Cat.# V4251) Ammonium Persulfate, 10% (Cat.# V3131) Urea (Cat.# V3171) bind silane (methacryloxypropyltrimethoxysilane) Gel Slick solution (Cambrex Cat.# 50640) 0.5% acetic acid in 95% ethanol Nalgene tissue culture filter (0.2 micron) polyacrylamide gel electrophoresis apparatus for gels 30cm (e.g., SA32 or S2) glass plates and side spacers for polyacrylamide gel 30cm 14 cm vinyl doublefine sharkstooth comb(s), 49 point, 0.4mm thick; or square-tooth comb, 35 cm, 60 wells (cut in half for 30 wells/gel), 0.4 mm thick (Owl Scientific Cat.# S2S-60A) power supply Liqui-Nox detergent (Use of Liqui-Nox detergent is extremely important, because other kinds of detergent can build up on the glass plates.) clamps (e.g., large office binder clips) diamond pencil for marking glass plates Page 12

13 Notes 1. Use a 4% gel for separation of the CTTv and amelogenin loci. 2. Unpolymerized acrylamide is a neurotoxin and suspected carcinogen; avoid inhalation and contact with skin. Read the warning label, and take the necessary precautions when handling this substance. Always wear gloves and safety glasses when working with acrylamide powder or solutions. 3. Bind silane is toxic and should be used in a chemical fume hood. 4. The longer glass plate will be treated with Gel Slick solution to prevent the gel from sticking, and the shorter glass plate will be treated with bind silane to bind the gel. The two plates must be kept apart at all times to prevent cross-contamination. 5. All cleaning utensils (sponges) for the longer glass plates should be kept separate from those for the shorter glass plates to prevent cross contamination of the binding solution. 6. The shorter glass plate preparation must be repeated for each gel. The longer glass plate preparation must be repeated after every four gels. 7. To remove the glass plate treatments (Gel Slick solution or bind silane) immerse the plate(s) in 10% NaOH solution for 1 hour. Thoroughly rinse the plate(s) with deionized water, and clean with a detergent. The same 10% NaOH solution may be used for multiple gels. 8. New glass plates should be soaked in 10% NaOH for 1 hour, then rinsed thoroughly with deionized water before use. New plates also should be etched with a diamond pencil in the corner of one side to distinguish the sides of the plates in contact with the gel. Page 13

14 Procedure Note: See comments at the end of this section for information on precast polyacrylamide gels. The following protocol is for the preparation of a denaturing polyacrylamide gel with the dimensions of 31.0 cm wide 38.5 cm high 0.4 mm thick (e.g., S2 sequencing gel electrophoresis apparatus, Whatman Cat.# ). Use one-half of the volumes described here for a gel with the dimensions of 17 cm wide 32 cm high 0.4 mm thick (e.g., SA32 sequencing gel apparatus, Whatman Cat.# ). 1. Thoroughly clean the shorter and longer glass plates twice with 95% ethanol and Kimwipes tissues. Note: The gel side is the etched side of the glass plate. 2. Using gloves, apply 3 ml of Gel Slick solution onto the etched side of the longer glass plate. With a dry paper towel, spread the Gel Slick solution using a circular motion over the entire surface. 3. Wait 5 minutes for the Gel Slick solution to dry. Remove the excess Gel Slick solution with a paper towel saturated with deionized water. Finally, dry the glass plate with Kimwipes tissue. 4. In a chemical fume hood, prepare fresh binding solution by adding 3 µl of bind silane to 1 ml of 0.5% acetic acid in 95% ethanol in a 1.5 ml tube. Wipe the etched side of the shorter glass plate using a Kimwipes tissue saturated with the freshly prepared binding solution. Be certain to wipe the entire plate surface with the saturated tissue. 5. Wait 5 minutes for the binding solution to dry. Wipe the shorter glass plate 3 4 times with 95% ethanol and Kimwipes tissues to remove the excess binding solution. Failure to wipe excess binding solution from the shorter glass plate will cause the gel to stick to both plates, and the gel will be destroyed upon separation of the glass plates after electrophoresis. 6. Take special care not to allow the treated surfaces to touch each other. Assemble the glass plates by placing 0.4 mm side spacers and a 0.4 mm bottom spacer (optional) between the plates and using clamps to hold them in place. Lean the assembled plates against a test tube rack or other similar support. 7. Prepare a 4% acrylamide solution (total of 75 ml) by combining the ingredients listed below: Urea g deionized water ml TBE Buffer, 10X 3.75 ml 40% acrylamide:bis (19:1) 7.50 ml total volume 75 ml 8. Filter the acrylamide solution through a 0.2 micron filter (e.g., Nalgene tissue culture filter). 9. Pour the filtered acrylamide solution into a squeeze bottle. 10. Add 50 µl of TEMED and 500 µl of 10% ammonium persulfate to the acrylamide solution, and mix gently. Page 14

15 11. Carefully pour the acrylamide solution between the glass plates. To prevent bubble formation, start pouring at one side of the assembled plates and maintain a constant flow of solution. 12. Position the gel horizontally, resting it on two test tube racks or other similar supports. Remove any bubbles that may have formed. 13. Insert one or two 14 cm doublefine (49 point) sharkstooth combs, straight side into the gel, between the glass plates (6 mm of the comb should be between the two glass plates). If using a square-tooth comb, insert the comb between the glass plates until the teeth are almost completely inserted into the gel. 14. Secure the comb(s) with 2 to 3 clamps each. 15. Pour the remaining acrylamide solution into a disposable conical tube as a polymerization control. Rinse the squeeze bottle, including the spout, with water. 16. Allow polymerization to proceed for at least 1 hour. Check the polymerization control to be sure that polymerization has occurred. Note: The gel may be stored overnight if a paper towel saturated with deionized water and plastic wrap are placed around the well end of the gel to prevent the gel from drying out. If no bottom spacer is used, the bottom of the gel should be wrapped. III.D. Polyacrylamide Gel Electrophoresis Gel Pre-Run 1. Remove the clamps from the polymerized acrylamide gel, and clean the glass plates with paper towels saturated with deionized water. 2. Shave any excess polyacrylamide away from the comb. Remove the comb and bottom spacer. 3. Add 0.5X TBE to the bottom chamber of the electrophoresis apparatus. 4. Gently lower the gel and glass plates into the buffer with the longer plate facing out and the well side on top. 5. Secure the glass plates to the sequencing gel apparatus. 6. Add 0.5X TBE to the top buffer chamber of the electrophoresis apparatus. 7. Using a cc syringe filled with buffer, remove the air bubbles on the top of the gel. Be certain the well area is devoid of air bubbles and small pieces of polyacrylamide. Use a syringe with a bent 19-gauge needle to remove the air bubbles between the glass plates on the bottom of the gel. 8. Pre-run the gel to achieve a gel surface temperature of approximately 50 C. Consult the manufacturer s instruction manual for the recommended electrophoresis conditions. Note: As a reference, we generally use watts for a 40 cm polyacrylamide gel watts for a 32 cm gel. The gel running conditions may have to be adjusted to reach a temperature of 50 C. Page 15

16 Sample Preparation 1. Prepare the PCR samples by mixing 2.5 µl of each sample with 2.5 µl of STR 2X Loading Solution. Note: The sample alleles may appear more intense than ladder alleles on the gel, but this should not interfere with allele determination. For more even band intensities, mix 1 µl of each sample with 4 µl of a premix containing 2.5 µl of STR 2X Loading Solution and 1.5 µl of STR 1X Buffer. 2. Add 2.5 µl (50 ng) of pgem DNA Markers to 2.5 µl of STR 2X Loading Solution for each marker lane. Note: We recommend loading pgem DNA Markers into the first and last lanes of the gel. 3. Combine 2.0 µl of the CTT Allelic Ladder and 2.0 µl of Amelogenin ladder. Mix well then combine 2.5 µl of this mixture with 2.5 µl of STR 2X Loading Solution for each allelic ladder lane. The number of allelic ladder lanes used depends on personal preference. 4. Briefly centrifuge the samples in a microcentrifuge to bring the contents to the bottom of the tube. Sample Loading 1. Denature the samples by heating at 95 C for 2 minutes, then immediately chill on crushed ice or in an ice-water bath. Note: Denature the samples just prior to loading the instrument. 2. After the pre-run, use a cc syringe filled with buffer to flush the urea from the well area. If using a sharkstooth comb, carefully insert the comb teeth into the gel approximately 1 2 mm. Leave the comb inserted in the gel during both gel loading and electrophoresis. 3. Load 3 µl of each sample into the respective wells. The loading process should take no longer than 20 minutes to prevent the gel from cooling. Gel Electrophoresis 1. Once loading is complete, run the gel using the same conditions as for the gel pre-run. Note: In a 4% gel, bromophenol blue migrates at approximately 40 bases and xylene cyanol migrates at approximately 170 bases. 2. Knowing the size ranges for each locus (Table 4) and migration characteristics of the dyes (Step 1, above), stop electrophoresis any time after the locus of interest has passed the midpoint of the gel. If running more than one locus or a multiplex, be careful not to run the TH01 locus off the bottom of the gel. 3. Proceed to silver stain detection. Page 16

17 Precast Polyacrylamide Gels Because so many components of polyacrylamide gels are toxins, you may wish to use precast gels. Most precast gels that are sold specifically for separation of DNA fragments and silver stain detection are designed to run on a specific apparatus. Therefore, to use precast gels, you may need to also purchase an appropriate gel electrophoresis apparatus. Several biotechnology supply companies sell such systems. Invitrogen sells Novex precast DNA retardation gels, and they specify the size range that you can expect to separate when you use these gels. Their gels are designed to run on the XCell SureLock MiniCell apparatus. GE Life Sciences sells the GenePhor DNA Separation System and precast gels. III.E. Silver Staining This protocol describes silver staining of polyacrylamide gels. Materials to Be Supplied by the User SILVER SEQUENCE Staining Reagents (Cat.# Q4132) fix/stop solution staining solution developer solution (chilled to 4 10 C) Nalgene wash tubs ( cm or appropriate size for your system) orbital shaker or rocker platform fix/stop solution 10% glacial acetic acid staining solution 1 g/l silver nitrate (AgNO 3 ) 0.056% formaldehyde (HCOH) (1.5 ml of 37% HCOH/liter) developing solution 30 g/l sodium carbonate (Na 2 CO 3 ) M formaldehyde (HCOH)(1.5 ml of 37% HCOH/liter) 2 mg/l sodium thiosulfate(na 2 S 2 O 3 5H 2 O) Use 2 liters of each solution per gel for each step (for a cm tray). Procedure 1. After electrophoresis, empty the buffer chambers and carefully loosen the gel clamps. Remove the glass plates from the apparatus. 2. Place the gel and glass plates on a flat surface. Remove the comb and side spacers. Use a plastic wedge to carefully separate the two glass plates. The gel should be strongly affixed to the shorter glass plate. 3. Place the gel (attached to the shorter plate) in a shallow plastic tray (e.g., Nalgene wash tub). 4. To silver stain, follow Steps a h below. Gently agitate during each step. Steps involving solutions containing formaldehyde should be performed in a chemical hood. Page 17

18 Step Solution Time a. fix/stop solution (See Note 1) 20 minutes b. deionized water 2 minutes c. repeat Step b, twice 2 2 minutes d. staining solution 30 minutes e. deionized water (See Note 2) 10 seconds f. developer solution up to 5 minutes (untill alleles and ladders are visible) g. fix/stop solution (See Note 3) 5 minutes h. deionized water 2 minutes Notes: 1. Save the fix/stop solution from Step 4a, to use in Step 4g. 2. The duration of Step 4e is important. The total time from immersion in deionized water to immersion in developer solution should be less than 20 seconds. If the deionized water rinse step does exceed 20 seconds, repeat Step 4d. 3. Add fix/stop solution directly to developer solution to stop developing reaction. 4. Position the gel and shorter plate upright, and allow it to dry overnight. For best results, the gel should be completely dried before APC Film development (Section III.F). Alternatively, to create film prints of the gel immediately, cover the gel with plastic wrap, and expose your film. Reusing Glass Plates 1. Immerse the plate and affixed gel in a 10% NaOH solution for 1 hour to overnight. Discard the gel, and clean the glass plate with deionized water and a detergent such as Liqui-Nox detergent. The 10% NaOH solution may be reused for additional gels. 2. All cleaning utensils and sponges for the longer glass plates should be kept separate from those for the shorter glass plates to prevent cross-contamination of the binding solution. Page 18

19 III.F. Generating Film Images A direct image may be produced using Automatic Processor Compatible (APC) Film. The image produced on APC Film is the mirror image of the gel. Use of film allows the generation of multiple permanent images with more control over band and background intensity than does development of the gel alone. Handle all plates with gloved hands to avoid fingerprints. Materials to Be Supplied by the User white light box automatic film processor or film developing tanks Automatic Processor Compatible (APC) Film (Cat.# Q4411) 1. In the darkroom with a safelight on, place the dry, stained gel attached to the shorter plate (gel side up) on a white fluorescent light box. Note: For best results, the gel should be completely dry before the image is captured with APC film. If capturing an image from a gel that has not been dried, cover the gel with plastic wrap. 2. Position the APC Film, emulsion side down, over the gel to be copied. Note: The emulsion side of the film can be identified as the glossy white surface; the nonemulsion side has a gray tint. 3. Place a clean glass plate on top of the film to maintain contact between the gel and film. Turn on the white light box, and expose the film for 1 2 minutes, depending on the gel background level and the intensity of the white light. (This step must be optimized for individual light boxes.) 4. Develop the film as recommended by the manufacturer. APC film may be processed manually or with an automatic film processor. For automatic film processors, follow the manufacturer s instructions. Note: The image produced on APC Film is the mirror image of the gel. 5. If there is very little signal, decrease the exposure time used in Step 3. If the film appears brown or black, increase the exposure time. Page 19

20 III.G. Analyzing Data Run your allelic ladders in lanes adjacent to each sample to ease interpretation of results. Direct comparison between the allelic ladders and amplified samples of the same locus allows easier assignment of alleles. The TH01 allele 9.3 is a microvariant allele and does not comigrate with allelic ladder fragments. In addition, mutations or rare alleles may be seen occasionally. The migration of such off-ladder alleles cannot be predicted. With silver stain detection, both DNA strands are detected. For some loci, such as TH01, the difference in the sequence of the opposing strands causes them to migrate at different rates. This results in doublets for each allele (Figure 1). This strand separation may be more pronounced with longer electrophoresis of gels. Artifact bands also may be detected with these systems. Shadow banding (1 3) or repeat slippage appears as faint bands one repeat unit (i.e., 4 bases) below the true alleles. Terminal nucleotide addition occurs when Taq DNA polymerase catalyzes template-independent addition of a nucleotide to the 3 -termini of amplified DNA fragments (3 5). A band that is one base shorter than the expected allele may result from the inefficiency of the terminal nucleotide addition. An artifact band is generated when this terminal addition does not occur with 100% efficiency. This may be visualized as an extra band. pgem DNA Markers The pgem DNA Markers are visual standards used to confirm allelic size ranges for the loci. The markers consist of fifteen DNA fragments with the following sizes (in base pairs): 2, , , Controls Observe the lanes containing the negative controls. They should be devoid of amplification products. Observe the lanes containing the positive K562 DNA positive controls. Compare the K562 DNA allelic repeat sizes with the locus-specific allelic ladder. The expected K562 DNA allele size(s) for each locus are listed in Table 4. STR Ladders Each locus or multiplex has a characteristic allelic ladder. Please refer to Table 4 for locus-specific allelic ladder information. In general, the allelic ladders contain fragments of the same lengths as either several or all known alleles for the locus. Visual comparison between the allelic ladder and amplified samples of the same locus allows precise assignment of alleles. Page 20

21 Representative Data CSF1PO TPOX CTT Multiplex L 1 2 L 3 4 L Figure 1. Representative FFv data. Individual SilverSTR genomic DNA III samples (lanes 1 4) Multiplex were amplified using GenePrint Multiplex STR Systems as indicated and L12L34L detected using silver staining as described in this manual. L 1 2 L 3 4 L The amplification products 16 were separated using a 4% denaturing 15 polyacrylamide gel. F13A01 D16S539 Numbers to the right of 4 each image indicate the smallest and largest number of repeat units present in corresponding fragments of 5the 14 allelic ladder. 14 FESFPS 7 D7S820 6 TH D13S vwa TA L L Amelogenin 218bp (Y) 212bp (X) 0753TM10_4B Figure 2. Amplification of varying concentrations of K562 template DNA at the Amelogenin locus. DNA was amplified using a Perkin-Elmer model 480 thermal cycler. Lanes 1 and 8 contain the locus-specific allelic ladder; lanes 2 6 contain amplified K562 DNA using 250, 25, 5, 1 and 0.5 ng of starting template,respectively. Table 3. Locus-Specific Information for CTT plus Amelogenin Multiplex. STR Locus Chromosomal Location GenBank Locus and Locus Definition Repeat Sequence 5 3 Amelogenin1 Xp and Y CSF1PO1 5q TH01 11p15.5 TPOX 2p25.1 pter HUMANEL, Human Y chromosomal gene for amelogenin-like protein NA HUMCSF1PO, Human c-fms proto-oncogene for CSF-1 receptor gene AGAT 2 HUMTH01,Human tyrosine hydroxylase gene AATG 2 HUMTPOX, Human thyroid peroxidase gene AATG2 1Amelogenin is not an STR, but displays a 212-base, X-specific band and a 218-base, Y-specific band. K562 DNA (female) displays only the 212-base, X-specific band. 2Repeat sequences represent all four possible permutations (e.g., AGAT is used for AGAT, GATA, ATAG or TAGA). The first alphabetic representation of the repeat (e.g., AGAT) is used according to the precedent of Edwards et al. (6). Page 21

22 Table 4. Additional Locus-Specific Information for the CTT plus Amelogenin Multiplex. STR Locus Allelic Ladder Size Range 1 (bases) STR Ladder Alleles (# of repeats)2 Other Known Alleles 3 (# of repeats) K562 DNA Allele Sizes (# of repeats) Comments Amelogenin NA None 212,212 1,2 CSF1PO ,8,9,10,11, 12,13,14,15 6 9,10 1 TH ,6,7,8,9,10, ,9.3 1,3 6,7,8,9,10,11, TPOX ,13 None 8,9 1 1Lengths of each allele in the allelic ladders have been confirmed by sequence analyses. 2Alleles in bold are present in greater amounts than other alleles. This simplifies interpretation. 3Alleles that represent <0.2% of the population may not be listed in this table. 4Amelogenin is not an STR, but displays a 212-base X-specific band and a 218-base Y-specific band. K562 DNA (female) displays only the 212-base X-specific band. Comments 1. PCR amplification sometimes generates artifacts that appear as faint bands below the alleles. These products probably result from a process known as slippage, commonly observed in PCR amplification of regions that contain tandem repeats of short sequences (1 3). 2. A strong extra band may be observed below the 212 bp Amelogenin allele when more than 25 ng of template DNA is amplified. 3. Locus TH01 has a common 9.3 allele (7). A one-base deletion is present in the allele that contains 10 repeats. Note that reference 6 refers to this allele as 10.1 rather than 9.3. This allele was renamed 9.3 at the ISFH Conference in Venice, Italy, in October Power of Discrimination The following tables provide information about the power of discrimination (matching probability, paternity index [PI], and power of exclusion) within a variety of populations using the alleles in the CTT Multiplex (not including Amelogenin). A measure of discrimination often used in paternity analyses is the paternity index (PI), a means for presenting the genetic odds in favor of paternity given the genotypes for the mother, child and a tested man (8). An alternative calculation used in paternity analyses is the power of exclusion (8). Table 5. Population Statistics for the CTT Triplex. African-American Caucasian-American Hispanic-American Matching Probability 1 in 1,590 1 in in 549 Paternity Index Power of Exclusion Page 22

23 III.H. Obtaining Single-Source Human Genomic DNA STR mapping requires single-source human genomic DNA. You can have your students use the K562 DNA provided with the CTT Multiplex System. However, if you would like your students to compare two different DNA samples using STR mapping, human cultured cells can be used as an source for genomic DNA. There are many cell lines available from the American Type Culture Collection (ATCC), and the genotypes of lines supplied by ATCC are known. However, use of cell-line-derived DNA may result in allelic imbalance and imbalance between STR loci. To obtain DNA from tissue culture cells, use the Wizard SV Genomic DNA Purification System. This system uses either a spin or a vacuum protocol. Details about genomic DNA isolation using this kit are available in the Wizard SV Genomic DNA Purification System Technical Bulletin #TB302, available at: This protocol is also available in Unit 4 of the Education Resources Web site ( Note: Instructors are responsible for ensuring that your institution s guidelines regarding studentprovided samples and work with human biological materials are followed. Be sure you know and understand your institution s rules and regulations if you decide to include human samples in your genotyping exercise. Many institutions have restrictions regarding student-provided samples for teaching labs because of ethical and safety considerations. Often such teaching laboratories will be subject to review by an internal review board (IRB). Be sure that you thoroughly investigate and follow your institution s guidelines regarding student-provided samples and working with human biological material if you decide to have the students genotype their own DNA. The DNA IQ System (Cat.# DC6700) is a DNA isolation and quantitation system designed specifically for forensic and paternity samples (8). This novel system uses paramagnetic particles to prepare clean samples for STR analysis easily and efficiently. The DNA IQ Resin eliminates PCR inhibitors and contaminants frequently encountered in casework samples. With larger samples, the DNA IQ. System delivers a consistent amount of total DNA. The system has been used to isolate and quantify DNA from routine sample types including buccal swabs, stains on FTA paper and liquid blood. Additionally, DNA has been isolated from casework samples such as tissue, differentially separated sexual assault samples and stains on support materials. Page 23

24 IV. STR Analysis of DNA Using the PowerPlex 16 System Modern forensic and paternity labs analyze DNA samples using the multiplex amplification and capillary electrophoresis. However, this level of analysis requires significant capital investment, and the required equipment and facilities are not often available for teaching labs. However, instructors at large insitutions may have access to the required equipment through a core laboratory facility. The PowerPlex 16 System (10,11) allows co-amplification and three-color detection of sixteen loci (fifteen STR loci and Amelogenin), including Penta E, D18S51, D21S11, TH01, D3S1358, FGA, TPOX, D8S1179, vwa, Amelogenin, Penta D, CSF1PO, D16S539, D7S820, D13S317 and D5S818. One primer for each of the Penta E, D18S51, D21S11, TH01 and D3S1358 loci is labeled with fluorescein (FL); one primer for each of the FGA, TPOX, D8S1179, vwa and Amelogenin loci is labeled with carboxy-tetramethylrhodamine (TMR); and one primer for each of the Penta D, CSF1PO, D16S539, D7S820, D13S317 and D5S818 loci is labeled with 6-carboxy-4,5 - dichloro-2,7 -dimethoxy-fluorescein (JOE). All sixteen loci are amplified simultaneously in a single tube and analyzed in a single injection or gel lane. The PowerPlex 16 Monoplex System, Penta E (Fluorescein) (Cat.# DC6591) and PowerPlex 16 Monoplex System, Penta D (JOE) (Cat.# DC6651) are available to amplify the Penta E and Penta D loci, respectively. Each monoplex system allows amplification of a single locus to confirm results obtained with the PowerPlex 16 System. The monoplex systems can be also used to re-amplify DNA samples when one or more of the loci do not amplify initially due to nonoptimal amplification conditions or poor DNA template quality. The PowerPlex 16 System is compatible with the ABI PRISM 310, 3100 and Avant Genetic Analyzers, and Applied Biosystems 3130 and 3130xl Genetic Analyzers. If you would like to pursue a forensics laboratory using these state-of-the-art systems, please see the PowerPlex 16 System Technical Manual #TMD012 available at: Alternatively the BioPharmaceutical Center Institute (BTCI) in Fitchburg, Wisconsin, conducts on-site workshops. Visit their Web site: for more information. Page 24

25 V. Supplier and Ordering Information Please see the Materials Required list that preceeds each protocol for a list of required equipment, props and reagents required for the laboratory that you are performing. RFLP Laboratory Product XmnI HincII pgl4.11 [luc2p] Vector pgl4.12 [luc2cp] Vector Agarose, LE, Analytical Grade TBE Buffer, 10X BenchTop pgem DNA Markers Cat.# R7271 R6031 E6661 E6671 V3121 V4251 G7521 STR/Silver Staining Laboratory Product Nuclease-Free Water GoTaq DNA Polymerase Mineral Oil GenePrint SilverSTR CTT Multiplex GenePrint Sex Identification Amelogenin (Silver Detection) SILVERSEQUENCE Staining Reagents TBE Buffer, 10X Cat.# P1193 DY1151 DC6001 DC4081 Q4132 V4251 Page 25