PCR-based Alu-Human DNA Typing

Transcription

1 The Biotechnology Education Company Revised and Updated PCR-based Alu-Human DNA Typing Storage: See Page 3 for specific storage instructions Experiment Objective: The objective of this experiment is for students to isolate human DNA and compare DNA polymorphisms between individuals. This experiment is designed for DNA staining with InstaStain Ethidium Bromide. EDVOTEK, Inc EDVOTEK

2 2 xxx Table of Contents PCR-based Alu-Human DNA Typing Experiment Components 3 Experiment Requirements 4 Background Information 5 Experiment Procedures Experiment Overview and General Instructions 9 Laboratory Safety 10 Module I-A: Isolation of DNA from Human Hair 11 Module I-B: Isolation of DNA from Human Cheek Cells 13 Module II: Amplification of the PV92 Locus 15 Module III: Separation of PCR Reactions by Electrophoresis 17 Study Questions 19 Instructor's Guidelines Notes to the Instructor 22 Pre-Lab Preparations 25 Experiment Results and Analysis 28 Study Questions and Answers 29 Appendices A PCR Experimental Success Guidelines 32 B Polymerase Chain Reaction Using Three Waterbaths 34 C Preparation and Handling of PCR Samples With Wax 35 D 1.0% Agarose Gel Preparation 36 E 1.0% Agarose Gels - Quantity Preparations 37 F Staining and Visualization of DNA with InstaStain Ethidium Bromide Cards 38 G InstaStain Blue: One Step Staining and Destaining 39 Material Safety Data Sheets 40 EDVOTEK, The Biotechnology Education Company, and InstaStain are registered trademarks of EDVOTEK, Inc.. Ready-to-Load and UltraSpec-Agarose are trademarks EDVOTEK - The Biotechnology of EDVOTEK, Education Inc. Company EDVOTEK

3 PCR-based Alu-Human DNA Typing 3 Components & Requirements Experiment # contains material for up to 25 human DNA typing reactions. Sample volumes are very small. For liquid samples, it is important to quick spin the tube contents in a microcentrifuge to obtain sufficient volume for pipeting. Spin samples for seconds at maximum speed. Storage A. Tubes with PCR reaction pellets Room Temperature Each PCR reaction pellet contains dntp Mixture Taq DNA Polymerase Buffer Taq DNA Polymerase MgCl 2 B. PV92 Primer mix -20 C Freezer C. 200 base pair ladder -20 C Freezer D. Control DNA -20 C Freezer E. Tris buffer -20 C Freezer F. Proteinase K Room temperature G. Chelating Agent Room temperature H. 10x PBS Room temperature Reagents & Supplies (Store all components below at room temperature) All components are intended for educational research only. They are not to be used for diagnostic or drug purposes, nor administered to or consumed by humans or animals. THIS EXPERIMENT DOES NOT CONTAIN HUMAN DNA. of the experiment components are derived from human sources. UltraSpec-Agarose Electrophoresis Buffer (50x) 10x Gel Loading Solution InstaStain Ethidium Bromide InstaStain Blue Conical tubes, 15 ml Microcentrifuge Tubes PCR tubes (0.2 ml - for thermal cyclers with 0.2 ml template) Cotton swabs Calibrated transfer pipets Wax beads (for waterbath option or thermal cyclers without heated lid) EDVOTEK - The Biotechnology Education Company EDVOTEK FAX: (301) info@edvotek.com

4 4 xxx PCR-based Alu-Human DNA Typing Requirements *If you do not have a thermal cycler, PCR experiments can be conducted, with proper care, using three waterbaths. However, a thermal cycler assures a significantly higher rate of success. Thermal cycler (EDVOTEK Cat. # 541 highly recommended) or three waterbaths* Horizontal gel electrophoresis apparatus D.C. power supply Balance Microcentrifuge Water bath (56 C) UV Transilluminator or UV Photodocumentation system UV safety goggles Automatic micropipets (5-50 µl) with tips Microwave, hot plate or burner Pipet pump 250 ml flasks or beakers Hot gloves Disposable vinyl or latex laboratory gloves Ice buckets and ice Distilled or deionized water Bleach solution Online Ordering now available Visit our web site for information about EDVOTEK s complete line of hands-on experiments for biotechnology and biology education. EDVO-TECH SERVICE EDVOTEK Mon - Fri ( ) 9 am - 6 pm ET Technical Service Department Mon - Fri 9:00 am to 6:00 pm ET FAX: (301) Web: edvotek@aol.com Please have the following information ready: Experiment number and title Kit lot number on box or tube Literature version number (in lower right corner) Approximate purchase date EDVOTEK -

5 PCR-based Alu-Human DNA Typing 5 Alu-Human DNA Typing The human genome consists of 2.9 billion base pairs of DNA. Of this total, only about 5% consists of exons which code for protein. Introns and other noncoding sequences make up the remainder; although some of these sequences may possess undiscovered functions, most appear to have none. Many of these noncoding sequences appear to be self-replicating and are repeated hundreds or thousands of times throughout the genome. These repetitive sequences have been termed "selfish" or "parasitic" DNA, as they often appear to possess no function except for their own reproduction. These repetitive elements account for more than 20 percent of the human genome. In 1979, it was discovered that human DNA contains a 300 base pair repetitive element. Copies of this element contain a recognition site for the restriction enzyme Alu I, and were subsequently named Alu elements. Although Alu elements have been found in exons, most exist in introns and other non-coding regions. When Alu sequences do insert into protein-coding regions, disruption of the gene usually results, often causing harm to the organism. Alu sequences replicate through an RNA intermediate which is copied into a double-stranded DNA segment called a retrotransposon. The details of this process are not well understood. The retrotransposon then inserts elsewhere in the genome. It is also theorized that most Alu sequences are incapable of replication and that only a small number of "master genes" are duplicated to form new elements. Background Information Although all humans (and other primates) possess hundreds of thousands of Alu elements, variations in their placement may occur. DNA sequences which vary between individuals are known as polymorphisms. For example, one person may possess an Alu insertion at a specific DNA locus, while another individual lacks that insertion. Furthermore, the insertion may be present or absent on each homologous chromosome and are called dimorphic. One such dimorphic Alu sequence is found on a section of chromosome 16 known as the PV92 locus. This section of DNA is 700 nucleotides in length. Insertion of the 300 base pair Alu element results in a length increase to 1000 base pairs. One may test whether a person possesses an Alu insertion at the PV92 locus by amplification of the locus using the polymerase chain reaction (PCR). If a person is homozygous for the insertion, a gel of the PCR product will result in a single band at 1000 base pairs (Figure 1A). If a person is heterozygous, i.e., possesses the insertion on one chromosome 16 homologue but not the other, two bands will be present following PCR. One band will be 700 base pairs and the other will be 1000 (Figure 1B). If a person lacks the insertion on either chromosome homologue, that person is said to possess the null genotype and PCR will result in only one band at 700 base pairs (Figure 1C).

6 6 PCR-based Alu-Human DNA Typing Alu-Human DNA Typing Paternal Primer 1 Paternal Primer 1 Background Information Maternal Figure 1A: Homozygous for Alu insertion (Lane B) Paternal Alu Alu Primer 2 Maternal Figure 1B: Heterozygous for Alu insertion (Lane C) Primer 2 Primer base pair ladder A B C D (Lane A) Alu Maternal Figure 1C: Homozygous for no Alu insertion (Lane D) Primer 2 Results of PCR Amplification of PV92 Locus 1200bp 1000bp 800bp 600bp 400bp 200bp Figure 1: PV92 Locus on Chromosome 16 Gel results are not drawn to scale. To purify DNA for this type of analysis, almost any tissue or body fluid (except urine) may be used. The most common sources of human DNA are hair and buccal cells (cheek cells). Cells to be tested must be treated (lysed) to release their DNA into solution. Following lysis, the cells are resuspended in a chelating agent, which removes cellular cations that inhibit PCR. PCR was invented in 1984 by Kary Mullis who was awarded a Nobel Prize for his work in The enormous utility of PCR is based on its ease of use and its ability to amplify DNA. The PCR process (Figure 2) uses an enzyme known as Taq polymerase. This enzyme is purified from a bacterium originally isolated from hot springs and is stable at very high temperatures. Also included

7 PCR-based Alu-Human DNA Typing 7 Alu-Human DNA Typing Target Sequence Cycle 1 = = = Separation of two DNA strands Primer 1 Primer 2 3' 3' 3' 3' 3' 3' Denature 94 C Anneal 2 primers 40 C - 65 C Background Information 3' 3' Extension 72 C Cycle 2 3' 3' 3' 3' 3' 3' Cycle 3 3' 3' 3' 3' 3' 3' 3' 3' Figure 2: Polymerase Chain Reaction

8 8 PCR-based Alu-Human DNA Typing Alu-Human DNA Typing in the PCR reaction mixture are two (15-30 nucleotide) synthetic oligonucleotides, known as "primers" and the extracted DNA template also known as the target DNA. The Experiment In the first step of the PCR reaction, the target complementary DNA strands are melted (separated from each other) at 94 C, while the Taq DNA polymerase remains stable. In the second step, known as annealing, the sample is cooled to 65 C to allow hybridization of the two primers to the two strands of the target DNA. In this experiment, the target is the PV92 locus in the extracted DNA. In the third step, the temperature is raised to 72 C and the Taq DNA polymerase adds nucleotides to the primers to synthesize the new complementary strands. These three steps - denaturation, annealing, and DNA synthesis - constitute one PCR "cycle". This process is typicality repeated for cycles, amplifying the target sequence exponentially (Figure 2). PCR is performed in a thermal cycler, which is programmed to rapidly heat, cool and maintain samples at designated temperatures for varying amounts of time. In this experiment, each student will extract his/her DNA from hair or cheek cells and amplify DNA at the PV92 locus by PCR. As a control, DNA purified from a cultured human cell line may be used. The PCR product(s) will then be examined on agarose gels to determine whether the student is homozygous (+/+), heterozygous (+/-), or null (-/-) for an Alu insertion at the locus. Objectives of this experiment are the isolation of human DNA and the comparison of DNA polymorphisms between individuals by PCR amplification and gel electrophoresis.

9 PCR-based Alu-Human DNA Typing 9 Experiment Overview and General Instructions Before you start the experiment 1. Read all instructions before starting the experiment. 2. If you will be conducting PCR using a thermal cycler without a heated lid, also read the Appendix entitled "Preparation and Handling PCR Samples with Wax ". 3. If you will be using three waterbaths to conduct PCR, read the two appendices entitled "Polymerase Chain Reaction Using Three Waterbaths" and "Handling samples with wax overlays". 4. Write a hypothesis that reflects the experiment and predict experimental outcomes. Experiment Objective: The Experiment The objective of this experiment is for students to isolate human DNA and compare DNA polymorphisms between individuals. Brief Description of Experiment: In this experiment, each student will extract his/her DNA from hair or cheek cells and amplify DNA at the PV92 locus by PCR. The PCR product(s) will then be examined on agarose gels to determine whether the student is homozygous (+/+), heterozygous (+/-), or null (-/-) for an Alu insertion at the locus. DNA purified from a cultured human cell line is included and may be used as a positive control. This experiment has three modules: I. Isolation of DNA II. PCR Amplification of the PV92 Locus III. Separation of PCR Reactions by Electrophoresis Gel specifications This experiment requires a gel with the following specifications: Recommended gel size: 7 x 14 cm (long tray) Number of sample wells required 6 Placement of well-former template: first set of notches Agarose gel concentration: 1.0%

10 10 PCR-based Alu-Human DNA Typing Laboratory Safety 1. Gloves and goggles should be worn routinely as good laboratory practice. The Experiment 2. Exercise extreme caution when working with equipment that is used in conjunction with the heating and/or melting of reagents. 3. Do not mouth pipet reagents - use pipet pumps. Wear gloves and safety goggles 4. Exercise caution when using any electrical equipment in the laboratory. Although electrical current from the power source is automatically disrupted when the cover is removed from the apparatus, first turn off the power, then unplug the power source before disconnecting the leads and removing the cover. Turn off power and unplug the equipment when not in use. 5. EDVOTEK injection-molded electrophoresis units do not have glued junctions that can develop potential leaks. However, in the unlikely event that a leak develops in any electrophoresis apparatus you are using, IM- MEDIATELY SHUT OFF POWER. Do not use the apparatus. 6. Special caution is required when handling human cheek cells. Swabs used for harvesting cheek cells should be soaked in a 15% bleach solution after the cells are suspended in the buffer. Swabs can be disposed in solid was e after soaking in the bleach solution. 7. Always wash hands thoroughly with soap and water after handling reagents or biological materials in the laboratory.

11 PCR-based Alu-Human DNA Typing Module I-A: Isolation of DNA from Human Hair (Preferred method) 11 This experiment can alternatively be performed using DNA isolated from cheek cells (see page 13). It is critical that there is a sufficient volume of cells to obtain enough DNA to yield positive DNA fingerprinting results. To maximize success, carefully read and follow all experimental instructions. WARNING! Use only screw-cap tubes when boiling for DNA isolation. Do not use snap-top tubes when boiling. 1. Isolate 3-4 hairs containing a sheath, a barrel-shaped structure (often white in color) encircling the shaft near the base of the hair (see figure at right). If necessary, sheaths can be cut from the remainder of the hair shaft. HUMAN HAIR Shaft Sheath Root The Experiment The preferred source is hairs from the eyebrows. They are short in length and can be readily placed in the bottom of the tube. 2. Place the hairs in the bottom of a 1.5 ml screw-cap tube. 3. Obtain the lysis solution from your instructor. This lysis solution contains 25 mm Tris-HCl ph 8.0, 10% chelating agent, 50 µg/ml proteinase K. The chelating agent removes Mg (required by DNA degrading nucleases and DNA polymerases). The small beads need to be suspended in the buffer prior to delivery to the cell suspension. Proteinase K will digest proteins found in solution. 4. Mix the lysis solution by vortexing or pipeting up and down. Before the chelating agent settles, quickly remove 150 µl and add it to the tube containing the hair. 5. Make sure the hair sheaths are completely submerged in solution and are not stuck on the sides of the tube. 6. Place the tube in a 56 C waterbath for 15 minutes. 7. Remove the tube from the waterbath and allow it to cool for 30 seconds. 8. Vortex the tube for 15 seconds.

12 12 PCR-based Alu-Human DNA Typing Module I-A: Isolation of DNA from Human Hair (Preferred method) 9. Check again that the hair sheaths are completely submerged in solution and are not stuck on the sides of the tube. 10. Place the tube in a float and place in boiling water for 10 minutes. The Experiment WARNING! Use only screw-cap tubes when incubating in the waterbath for DNA isolation. Do not use snap-top tubes. Boiling is required to obtain cell lysis. The DNA is not degraded by boiling and nucleases will not degrade the DNA because the Mg is chelated (trapped). 11. Remove the tube from water and cool on ice for 2 minutes. 12. Vortex the tube for 10 seconds. 13. Spin in a microcentrifuge for 30 seconds. Centrifuge the cell suspension carefully. If a microcentrifuge is not available, allow the chelating agent to settle in the tube for 3 minutes. 14. Carefully remove 50 µl of supernatant and transfer it to a clean 0.5 ml microcentrifuge tube. Discard the tube with the pellet. Transfer the DNA (supernatant) to a new microcentrifuge tube very carefully. It is the step prior to the PCR reaction. If any chelex beads (as few as a couple) are transferred, they can easily trap the Mg cation which is required by the Taq DNA polymerase as a cofactor for catalysis. 15. Place the tube containing the supernatant on ice. 16. Proceed with steps as outlined in Module II: Amplification of the PV92 locus. OPTIONAL STOPPING POINT The supernatant may be stored at -20 C until the experiment is continued.

13 PCR-based Alu-Human DNA Typing 13 Module I-B: Isolation of DNA from Human Cheek Cells This experiment can alternatively be performed using DNA isolated from hair (see page 11). A sufficient volume of cells is required to ensure that there is enough DNA to yield positive DNA fingerprinting results. To maximize success, carefully read and follow all experimental instructions. Special caution is required when handling human cheek cells. Swabs used for harvesting cheek cells should be soaked in a 15% bleach solution after the cells are suspended in the buffer. Swabs can then be disposed in solid was e after soaking in the bleach solution. 1. Obtain cells by swabbing the inside of the mouth with a cotton-tipped applicator. Twirl the applicator while vigorously swabbing both cheeks, between the gum line and under the tongue. 2. Place the cotton head in 2 ml of PBS (in a labeled 15 ml conical tube) and twirl back and forth vigorously for 30 seconds to dislodge cells. Press the cotton head against the walls of the conical tube to squeeze out as much liquid as possible. The Experiment 1 ml 0.5 ml 0.25 ml ~ 60 μl < 20 μl 3. Place the cotton-tippped swab in a beaker or container filled with 15% bleach solution to soak and disinfect. 4. Using a fresh applicator, repeat steps 1 and 2. Twirl the applicator to add cells to the same tube containing the 2 ml of PBS. 5. Place the second cotton-tippped swab in the 15% bleach solution to soak and disinfect. 6. With a calibrated transfer pipet, transfer 2 ml of the cells in PBS into a 2 ml screw-cap microtest tube. 7. Spin at g in a microcentrifuge (with appropriate counterbalance) for 1 minute. Important: Check to see that the buffer is clear (not cloudy) and that there is a visible white pellet (approximately 7-8 mm in diameter). If the buffer is cloudy with little or no pellet, spin the tube(s) for an additional seconds. If the buffer is clear with little or no pellet, obtain another swab and repeat steps Pipet off the supernatant, using care to avoid discarding the pellet. 9. Mix the tube of chelating agent by inverting it several times. Chelating agent is used to bind cations released by the cells that inhibit PCR. It removes Mg (required by DNA degrading nucleases and DNA polymerases). The small beads need to be suspended in the buffer prior to delivery to the cell suspension.

14 14 PCR-based Alu-Human DNA Typing Module I-B: Isolation of DNA from Human Cheek Cells, continued The Experiment WARNING! Make sure you are using a screw-cap tube for boiling your sample. Do not use snap-top tubes when boiling. 10. Continue to mix the chelating agent with a calibrated transfer pipet by pipeting up and down several times. Then quickly add 100 µl of the chelating agent to the tube containing the pellet. Alternatively, use a micropipet with a disposable tip to measure 100 µl of the chelating agent suspension. It may be necessary to cut the end of the tip to create a larger opening to allow the chelating agent to pass through more easily. 11. Resuspend the pellet in chelating agent by pipetting up and down several times, or by vortexing gently. Check to see that the pellet is fully suspended. 12. Lyse the cells by placing the tube in a boiling waterbath for 10 minutes. 13. Allow the tube to cool for 2 minutes. Vortex for 10 seconds or tap the tube vigorously to mix. 14. Spin the tube in a microcentrifuge for 2 minutes to pellet the cell debris, leaving DNA in the supernatant. 15. Transfer µl of the supernatant to a clean 0.5 ml tube, using care to avoid disturbing the pellet. Discard the tube containing the pellet. Transfer the DNA (supernatant) to the clean microcentrifuge tube very carefully. It is the step prior to the PCR reaction. If any chelex beads (as few as one or two) are transferred, they can easily chelate (trap) the Mg required by the Taq DNA polymerase as a cofactor for catalysis. Any carryover of chelex to the PCR reaction will not yield results. 16. Place the sample (supernatant) on ice. 17. Proceed with steps as outlined in Module II: Amplification of the PV92 locus. OPTIONAL STOPPING POINT The supernatant may be stored at -20 C until the experiment is continued.

15 PCR-based Alu-Human DNA Typing 15 Module II: Amplification of the PV92 Locus PCR Reaction: 1. Transfer the PCR Reaction pellet to the appropriate sized tube (e.g. 0.5 ml or 0.2 ml) for your thermal cycler. The PCR reaction pellet contains Taq DNA polymerase, the four deoxytriphosphates, Mg +2 and buffer. Sample volumes are very small. For liquid samples, it is important to quick spin the tube contents in a microcentrifuge to obtain sufficient volume for pipeting. Spin samples for seconds at maximum speed. 2. Label the tube containing the PCR reaction pellet with your initials. 3. Tap the reaction tube to assure the reaction pellet is at the bottom of the tube. 4. Add the following to the pellet: PV92 primer solution 20.0 µl Cell DNA (supernatant) 5.0 µl 5. Gently mix the PCR reaction tube and quickly spin it in a microcentrifuge to collect all the sample at the bottom of the tube. Make sure the PCR reaction pellet is completely dissolved. 6. If your thermal cycler is equipped with a heated lid, proceed directly to polymerase chain reaction cycling. If your thermal cycler does not have a heated lid, or if you are cycling manually with three water baths, add one wax bead to the tube before proceeding to polymerase chain reaction cycling. The Experiment Control Reaction (optional): One control reaction can be prepared for the entire class by a student or the instructor. 7. Transfer the PCR Reaction pellet to the appropriate sized tube (e.g. 0.5 ml or 0.2 ml) for your thermal cycler 8.. To prepare the PCR Control reaction, add the following to the pellet: PV92 primer solution 20.0 µl Control DNA 5.0 µl 9. Gently mix the control tube and quickly spin it in a microcentrifuge to collect all the sample at the bottom of the tube. Make sure the PCR reaction pellet is completely dissolved.

16 16 PCR-based Alu-Human DNA Typing Module II: Amplification of the PV92 Locus, continued 10. If your thermal cycler is equipped with a heated lid, proceed directly to polymerase chain reaction cycling. If your thermal cycler does not have a heated lid, or if you are cycling manually with three water baths, add one wax bead to the tube before proceeding to polymerase chain reaction cycling. The Experiment POLYMERASE chain reaction cycling 11. For automatic cycling, each student should place his/her PCR tube (and the optional control reaction) in the programmed thermal cycler. Follow the same cycling schedule if you are manually cycling in three water baths. Initial Denaturation 32 Final Extension 94 C for 5 min. 94 C for 30 sec. 72 C for 4 min. 61 C for 30 sec. 72 C for 45 sec. 12. After the cycles are completed, add 5 µl of 10x Gel Loading Solution to the sample and store on ice until ready for electrophoresis. 13. Proceed to instructions for preparing a 1.0% agarose gel (7 x 14 cm) and separating the PCR products by electrophoresis. OPTIONAL STOPPING POINT The samples can be held in the thermal cycler at 4 C or frozen after addition of 5 µl of 10x Gel Loading Solution until ready for electrophoresis.

17 PCR-based Alu-Human DNA Typing 17 Module III: Separation of PCR Reactions by Electrophoresis If you are unfamiliar with agarose gel preparation and electrophoresis, detailed instructions and helpful resources are available at Agarose Gel Requirements Recommended gel size: 7 x 14 cm 7 x 14 cm gels are recommended to achieve better resolution of the PCR products. Each gel can be shared by several students or groups. Placement of well-former template: first set of notches Agarose gel concentration: 1.0% Preparing the AGAROSE GEL 1. Close off the open ends of a clean and dry gel bed (casting tray) by using rubber dams or tape. 2. Place a well-former template (comb) in the first set of notches at the end of the bed. Make sure the comb sits firmly and evenly across the bed. The Experiment Important Note Continue heating until the final solution appears clear (like water) without any undissolved particles. Check the solution carefully. If you see "crystal" particles, the agarose is not completely dissolved. 3. To a 250 ml flask or beaker, add agarose powder and buffer as indicated in the Reference Tables (Appendix A) provided by your instructor. Swirl the mixture to disperse clumps of agarose powder. 4. With a marking pen, indicate the level of the solution volume on the outside of the flask. 5. Heat the mixture using a microwave oven or burner to dissolve the agarose powder. 6. Cool the agarose solution to 60 C with careful swirling to promote even dissipation of heat. If detectable evaporation has occurred, add distilled water to bring the solution up to the original volume marked in step 4. After the gel is cooled to 60 C: 7. Place the bed on a level surface and pour the cooled agarose solution into the bed. 8. Allow the gel to completely solidify. It will become firm and cool to the touch after approximately 20 minutes. 9. After the gel is solidified, be careful not to damage or tear the wells while removing the rubber dams or tape and comb(s) from the gel bed. 10. Place the gel (on its bed) into the electrophoresis chamber, properly oriented, centered and level on the platform. 11. Fill the electrophoresis apparatus chamber with the appropriate amount of diluted (1x) electrophoresis buffer (refer to Table B on the instruction Appendix provided by your instructor).

18 18 PCR-based Alu-Human DNA Typing Module III: Separation of PCR Reactions by Electrophoresis This experiment requires a 1.0% agarose gel and is designed for staining with InstaStain Ethidium Bromide. Loading DNA Samples The Experiment Reminder: Before loading the samples, make sure the gel is properly oriented in the apparatus chamber. - Black Sample wells + Red 1. (Optional Step) Heat the 200 bp DNA ladder and PCR samples for two minutes at 50 C. Allow the samples to cool for a few minutes. 2. Make sure the gel is completely submerged under buffer before loading the samples. Load the entire volume (30 µl) of the samples in the following sequence. Lane bp DNA ladder 2 Control DNA (optional) 3 Student #1 4 Student #2 5 Student #3 6 Student #4 3. Record the position of your sample in the gel for easy identification after staining. Running the Gel 4. After the DNA samples are loaded, properly orient the cover and carefully snap it onto the electrode terminals. 5. Insert the plugs of the black and red leads into the corresponding inputs of the power source. 6. Set the power source at the required voltage and conduct electrophoresis for the length of time determined by your instructor. 7. Check to see that current is flowing properly - you should see bubbles forming on the two platinum electrodes. 8. After the electrophoresis is completed, disconnect the power and remove the gel from the bed for staining. Staining and visualization of DNA After electrophoresis, agarose gels require staining to visualize the separated DNA samples. Your instructor will provide instructions for DNA staining with InstaStain Ethidium Bromide.

19 PCR-based Alu-Human DNA Typing 19 Study Questions Answer the following study questions in your laboratory notebook or on a separate worksheet. 1. Compare your Alu genotype with those of your classmates. Did anyone else have a similar result? If so, what are some possible explanations? 2. What is "selfish DNA"? How are Alu elements thought to replicate? What is the function(s) of Alu elements? 3. Could dimorphic Alu elements be used for DNA identification (i.e., in criminal investigations)? Why or why not? The Experiment

20 20 xxx PCR-based Alu-Human DNA Typing EDVOTEK -

21 PCR-based Alu-Human DNA Typing 21 Instructor s Guide Class size, length of laboratory sessions, and availability of equipment are factors which must be considered in the planning and the implementation of this experiment with your students. These guidelines can be adapted to fit your specific set of circumstances. If you do not find the answers to your questions in this section, a variety of resources are continuously being added to the EDVOTEK web site. In addition, Technical Service is available from 9:00 am to 6:00 pm, Eastern time zone. Call for help from our knowledgeable technical staff at EDVOTEK ( ). National Content and Skill Standards By performing this experiment, students will learn to load samples and run agarose gel electrophoresis. Analysis of the experiments will provide students the means to transform an abstract concept into a concrete explanation. Please visit our website for specific content and skill standards for various experiments. EDVO-TECH SERVICE EDVOTEK Mon - Fri ( ) 9 am - 6 pm ET Technical Service Department Mon - Fri 9:00 am to 6:00 pm ET FAX: (301) Web: edvotek@aol.com Please have the following information ready: Experiment number and title Kit lot number on box or tube Literature version number (in lower right corner) Approximate purchase date EDucational resources Electrophoresis Hints, Help and Frequently Asked Questions EDVOTEK experiments are easy to perform and designed for maximum success in the classroom setting. However, even the most experienced students and teachers occasionally encounter experimental problems or difficulties. The ED- VOTEK web site provides several suggestions and reminders for conducting electrophoresis, as well as answers to frequently asked electrophoresis questions. Online Ordering now available Visit our web site for information about EDVOTEK s complete line of hands-on experiments for biotechnology and biology education. EDVOTEK - The Biotechnology Education Company EDVOTEK FAX: (301) info@edvotek.com

22 22 PCR-based Alu-Human DNA Typing Notes to the Instructor: pcr Experimental Success Guidelines Please refer to the Appendices section for a summary of important hints and reminders which will help maximize successful implementation of this experiment. This experiment has three modules: The Experiment I. Isolation of DNA II. PCR Amplification of the PV92 Locus III. Separation of PCR Reactions by Electrophoresis Micropipetting Basics and Practice Gel Loading Accurate pipeting is critical for maximizing successful experiment results. EDVOTEK Series 300 experiments are designed for students who have had previous experience with agarose gel electrophoresis and micropipeting techniques. If your students are unfamiliar with using micropipets, EDVOTEK highly recommends that students perform Experiment # S-44, Micropipetting Basics, or other Series 100 or 200 electrophoresis experiment prior to conducting this advanced level experiment. Table C Volts APPROXIMATE TIME REQUIREMENTS 1. The PCR step (32 cycles) will take about minutes or can be processed overnight and held at 4 C. 2. The experiment can be temporarily stopped after the completion of Modules I and II and later resumed. Experimental results will not be compromised if instructions are followed as noted under the heading Optional Stopping Point at the end of Module I and Module II. 3. Whether you choose to prepare the gel(s) in advance or have the students prepare their own, allow approximately minutes for this procedure. Generally, 20 minutes of this time is required for gel solidification. See section Options for Preparing Agarose Gels Time and Voltage below. Recommended Time Minimum Maximum 55 min 2 hrs 15 min 3 hrs 25 min (1.0% - 7 x 14 cm gel) 1 hr 15 min 3 hrs 5 hrs 4. The approximate time for electrophoresis will vary from 1-5 hours. Generally, the higher the voltage applied, the faster the samples migrate. However, depending upon the apparatus configuration and the distance between the two electrodes, individual electrophoresis units will separate DNA at different rates. Follow manufacturer's recommendations. Time and Voltage recommendations for EDVOTEK equipment are outlined in Table C.

23 PCR-based Alu-Human DNA Typing 23 Notes to the Instructor: options for Preparing AGAROSE gels This experiment is designed for DNA staining after electrophoresis with InstaStain Ethidium Bromide. There are several options for preparing agarose gels for the experiment. 1. Individual Gel Casting: Each student lab group can be responsible for casting their own individual gel prior to conducting the experiment. 2. Preparing Gels in Advance: Gels may be prepared ahead and stored for later use. Solidified gels can be stored under buffer in the refrigerator for up to 2 weeks. Do not store gels at -20 C. Freezing will destroy the gels. Gels that have been removed from their trays for storage, should be "anchored" back to the tray with a few drops of hot, molten agarose before placing the gels into the apparatus for electrophoresis. This will prevent the gels from sliding around in the trays and the chambers. Instructor s Guide 3. Batch Gel Preparation: A batch of agarose gel can be prepared for sharing by the class. To save time, a larger quantity of UltraSpec-Agarose can be prepared for sharing by the class. See instructions for "Batch Gel Preparation". Gel concentration and volume The gel concentration required for this experiment is 1.0%. Prepare gels according to Table A.1 or A.2 in Appendix D.

24 24 PCR-based Alu-Human DNA Typing Notes to the Instructor: Gel Staining and Destaining AFTER ELECTROPHORESIS Instructor s Guide After electrophoresis, the agarose gels require staining in order to visualize the separated DNA samples. This experiment features a proprietary stain called InstaStain. InstaStain EtBr (Appendix F) Optimal visualization of PCR products on gels of 1.0% or higher concentration is obtained by staining with InstaStain Ethidium Bromide (InstaStain EtBr) cards. Exercise caution when using Ethidium Bromide, which is a listed mutagen. Disposal of the InstaStain EtBr cards, which contain only a few micrograms of ethidium bromide, is minimal compared to the large volume of liquid waste generated by traditional ethidium bromide staining procedures. Disposal of InstaStain cards and gels should follow institutional guidelines for chemical waste. InstaStain Blue: One-step Staining and Destaining (Appendix G) InstaStain Blue can be used as an alternative for staining gels in this experiment. However, InstaStain Blue is less sensitive than InstaStain EtBr and will yield variable results. Agarose gels can be stained and destained in one easy step, which can be completed in approximately 3 hours, or can be left in liquid overnight. For the best photographic results, leave the gel in liquid overnight. This will allow the stained gel to "equilibrate" in the destaining solution, resulting in dark blue DNA bands contrasting against a uniformly light blue background. Gels stained with InstaStain Blue may be stored in the refrigerator for several weeks. Place the gel in a sealable plastic bag with destaining liquid. DO NOT FREEZE AGAROSE GELS! Used InstaStain Blue cards and destained gels can be discarded in solid waste disposal. Destaining solutions can be disposed down the drain. Photodocumentation of DNA (Optional) There are many different photodocumentation systems available, including digital systems that are interfaced directly with computers. Specific instructions will vary depending upon the type of photodocumentation system you are using.

25 PCR-based Alu-Human DNA Typing 25 Pre-Lab Preparations: Module 1-A (Hair) 1. Add 100 µl Tris buffer (E) to the tube of Proteinase K (F) and allow the sample to hydrate for several minutes. After the sample is hydrated, pipet up and down several times to thoroughly mix the material. You may want to cut the end of the pipet tip to avoid clogging of the pipet with beads in the chelating agent. 2. Prepare the Lysis Solution: Transfer the entire amount of Proteinase K solution to the tube containing the chelating agent (G). Add an additional 4 ml of Tris buffer (E) to the chelating agent and invert the tube several times to obtain a uniform suspension. Quickly dispense 300 µl for each student pair. Cap and mix chelating agent in between each aliquot. Label these 13 tubes lysis solution. Instructor s Guide 3. Thaw the PV92 primer solution (B) and place on ice. Dispense 50 µl for each pair of students. Label these 13 tubes PV92 Primer. 4. Dispense 20 µl of 10x Gel Loading Solution for each student pair. 5. To each student, distribute the items as listed in the box below. Certain reagents are to be shared by two students. 6. There is also material for one control PCR reaction. You may wish to perform the control reaction in your thermal cycler before the students try their reactions. 7. Aliquot 30 µl of the 200 base-pair ladder (C) into microcentrifuge tubes. Distribute one tube of ladder per gel. Each Student should receive: One tube containing a PCR bead One 1.5 ml screw-cap microtest tube One calibrated transfer pipet One 0.5 ml microtest tube Reagents to be Shared by Two Students: Warning!! Remind students to only use screw-cap tubes when boiling DNA isolation samples. The snap-top tubes can potentially pop open and cause injury. 300 µl Lysis Solution 50 µl PV92 Primer Solution 20 µl 10x Gel Load Solution

26 26 PCR-based Alu-Human DNA Typing Pre-Lab Preparations Module I-B (Cheek Cells) 1. Pour the entire content of 10x PBS (H) into a 100 ml graduated cylinder. Dilute to 100 ml with distilled water and mix. Instructor s Guide 2. Add 4 ml of Tris buffer (E) to the tube containing the chelating agent (G). Invert the tube several times to obtain a uniform suspension and quickly dispense 300 µl for each student pair. Cap and mix chelating agent in between each aliquot. Label these 13 tubes "chelating agent". 3. Thaw the PV92 primer solution and place on ice. Dispense 50 µl for each pair of students. Label these 13 tubes PV92 Primer. 4. Dispense 20 µl of 10x Gel Loading Solution for each student pair. 5. To each student, distribute the items as listed in the box below. Certain reagents are to be shared by two students. 6. There is also material for one control PCR reaction. You may wish to perform the control reaction in your thermal cycler before the students try their reactions. 7. Aliquot 30 µl of the 200 base-pair ladder (C) into microcentrifuge tubes. Distribute one tube of ladder per gel. 8. For each pair of students, prepare small beakers or waste containers filled with 15% bleach solution for soaking cotton swabs after harvesting cheek cells. Each Student should receive: Warning!! Remind students to only use screw-cap tubes when boiling DNA isolation samples. The snap-cap tubes can potentially pop open and cause injury. Two (2) cotton-tipped swab applicators One tube containing a PCR bead One 15 ml conical tube containing 2 ml of 1x PBS. One 0.5 ml microtest tube Two calibrated transfer pipets One 2 ml screw-cap tube Reagents to be Shared by Two Students: 300 µl Chelating agent 50 µl PV92 Primer Solution 20 µl 10x Gel Load Solution 15% bleach solution

27 PCR-based Alu-Human DNA Typing 27 Pre-Lab Preparations Module II - Amplification of the PV92 locus The Thermal cycler should be programmed for the following cycles. Initial Denaturation 32 Final Extension 94 C for 5 min. 94 C for 30 sec. 72 C for 4 min. 61 C for 30 sec. 72 C for 45 sec. Accurate temperatures and cycle times are critical. A pre-run for one cycle (which will take approximately 3 to 5 min) is recommended to check that the thermal cycler is properly programmed. For thermal cyclers which do not have a top heating plate, it is necessary to place a layer of wax above the PCR reactions in the microcentrifuge tubes to prevent evaporation. Instructor s Guide Three water baths can be used for PCR if a thermal cycler is unavailable. The experiment will work if given great care and patience. Samples will require wax layers. Notes and Reminders: Accurate temperatures and cycle times are critical for PCR. A pre-run for one cycle (approx. 3 to 5 minutes) is recommended to check that the thermal cycler is properly programmed. For thermal cyclers that do not have a top heating plate, it is necessary to place a layer of wax above the PCR reactions in the microcentrifuge tubes to prevent evaporation. See Appendix entitled "Preparation and Handling PCR Samples with Wax ". Three water baths can be used for PCR if a thermal cycler is unavailable. The experiment will require great care and patience. Samples will require wax layers. See appendices entitled "Polymerase Chain Reaction Using Three Waterbaths" and "Handling samples with wax overlays". Module III - Separation of PCR Reactions by Electrophoresis Students will share gels in this experiment. Each 1.0% gel should be loaded with the 200 base pair ladder and samples from 4 or 5 students. The control PCR reaction can also be loaded in one of the wells.

28 28 PCR-based Alu-Human DNA Typing Experiment Results and Analysis Instructor s Guide 1200bp 1000bp 800bp 600bp 200 base pair ladder Student #1 Student #2 Student # bp 1000 bp 800 bp 600 bp 400 bp 200 bp 200 base pair ladder Student #1 Student #2 Student #3 400bp 200bp Short gel Long Gel Idealized Schematics The idealized schematics show the possible PCR products from different genotypes. Three possible results for PV92 amplification (shown above): Lane 1 Student #1 Homozygous for Alu insertion (+/+) Lane 2 Student #2 Heterozygous for Alu insertion (+/-) Lane 3 Student #3 No genotype for Alu insertion (-/-) Note: Depending on the PCR conditions used, a diffuse, smallmolecular weight band, known as a "primer dimer", may be present below the 200 bp marker (not observed in photo shown). This is a PCR artifact and can be ignored. Other minor bands may also appear due to nonspecific primer binding and the subsequent amplification of these sequences. Photo of Gel Results

29 PCR-based Alu-Human DNA Typing 29 Study Questions and Answers 1. Compare your Alu genotype with those of your classmates. Did anyone else have a similar result? If so, what are some possible explanations? Identical Alu genotypes at the PV92 locus indicate similarities in ancestry. It is extremely unlikely that Alu insertions would occur at the exact same location by coincidence. The null genotype, however, does not necessarily indicate common ancestry. 2. What is "selfish DNA"? How are Alu elements thought to replicate? What is the function(s) of Alu elements? Selfish DNA refers to DNA sequences that appear to exist for no other purpose than their own replication. These sequences, might, however, possess yet undiscovered functions beneficial to their host. Alu elements are theorized to be copied from a small number of "master genes", while the vast majority of elements are incapable of replication. These master genes replicate through a poorly-understood process called retrotransposition, in which the elements are transcribed to RNA, which is then reverse transcribed to double-stranded DNA. The newlyformed DNA copy ("retrotransposon") can then insert at a new, possibly random location in the genome. Instructor s Guide The function of Alu elements is not known. It may be that only a small number have function(s), while the majority of duplicated sequences serve no useful purpose. 3. Could dimorphic Alu elements be used for DNA identification (i.e., in criminal investigations)? Why or why not? Polymorphic Alu elements could not be used for DNA identification purposes, unless they are extremely rare in the population. These might be useful, however, for an initial screening to "narrow" possible identification to a small number of individuals.

30 30 xxx PCR-based Alu-Human DNA Typing EDVOTEK -

31 PCR-based Alu-Human DNA Typing 31 Appendices A B C D E F G PCR Experimental Success Guidelines Polymerase Chain Reaction Using Three Waterbaths Preparation and Handling of PCR Samples With Wax 1.0% Agarose Gel Preparation 1.0% Agarose Gels - Quantity Preparations Staining and Visualization of DNA with InstaStain Ethidium Bromide Cards InstaStain Blue: One Step Staining and Destaining Material Safety Data Sheets EDVOTEK - The Biotechnology Education Company EDVOTEK FAX: (301) info@edvotek.com

32 32 Appendix A PCR Experimental Success Guidelines EDVOTEK experiments which involve the extraction and amplification of DNA for fingerprinting are extremely relevant, exciting and stimulating classroom laboratory activities. These experiments have been performed successfully in many classrooms across the country, but do require careful execution because of the small volumes used. The following guidelines offer some important suggestions, reminders and hints for maximizing success. DNA Extraction and sample preparation: 1. Sufficient Cells: It is critical that there are sufficient cells to obtain enough DNA that will yield positive DNA fingerprinting results. Cell sources include human, plant, drosophila and bacterial cells. Without enough cells, there will not be enough DNA template for the PCR reaction. 2. Human (Self) DNA Fingerprinting: Cells obtained from human sources, such as cheek cells, need to be harvested cautiously. Aerosol can be a health hazard and cause cross-contamination among students. Hair follicles do not pose the same problem and yield sufficient DNA for the PCR reaction. HUMAN HAIR Shaft Sheath Root 3. Hair Cells: At least four (4) hair follicles are needed. The preferred source is hair from eyebrows. Use only hairs containing a sheath, a barrel-shaped structure (often white in color) encircling the shaft near the base of the hair (see figure at left). Centrifuge the hair follicles to the bottom of the microcentrifuge tube to ensure direct contact with the reagents used. 4. Cheek Cells: A white pellet must be visible after centrifugation of the cell suspension obtained from cheek swabbing. If necessary, repeat centrifugation to obtain a visible pellet. After removing the supernatant, suspend the pellet in chelating agent by repeated vortexing and pipetting up and down. 5. Chelating Agent: Chelating agent removes Mg (required by DNA-degrading nucleases and DNA polymerases). The small beads must be suspended in the buffer prior to delivery to the cells (i.e., mix the chelating agent just before you transfer it to the tube containing the cells. 6. Boiling: The boiling step for 10 minutes is required to obtain cell lysis. Boiling will not degrade the DNA and nucleases will NOT degrade DNA in the absence of Mg. 7. Centrifugation: Centrifuge the cell suspension carefully after cooling. If the pellet loosens, repeat this step. The supernatant should be clear, not cloudy, and the pellet should be solid at the bottom of the tube. Repeat centrifugation for a longer period of time, if necessary. Important Note Remember: Any carryover of chelating agent to the PCR reaction will not yield results. 8. DNA Transfer: Transfer the DNA to a new microcentrifuge tube very carefully. It is the step prior to the PCR reaction. If any chelating agent beads (as few as one or two) are transferred, they can easily trap the Mg required by the Taq DNA polymerase as a cofactor for catalysis. As an additional precaution, centrifuge the supernatant a second time.

33 Appendix A PCR Experimental Success Guidelines (continued) 33 The PCR Reaction 9. Add Primers and DNA to the PCR Reaction Bead: Add the primer mixture (forward and reverse primers) and the cell DNA (supernatant) as specified in the experimental procedures to the microcentrifuge tube containing the PCR reaction bead. Make sure that the bead (which contains the Taq DNA polymerase, the 4XdTPs, Mg and the PCR reaction buffer) is completely dissolved. Do a quick spin in a microcentrifuge to bring the entire sample to the bottom of the tube. Prepare the control reaction similarly. 10. The Thermal cycler: The thermal cycler must be programmed for the correct cycle sequence. It is critical that the temperatures and the time for each of the cycles are accurate. 11. Oil or Wax: For thermal cyclers that do not have a top heating plate, the reaction in the tubes must be overlaid with oil or wax to prevent evaporation. 12. Manual Water Bath PCR: Three water baths can be used as an alternative to a thermal cycler for PCR, but results are more variable. Samples require oil or wax layers. This method requires extra care and patience. Gel Preparation and Staining 13. Concentrated agarose: Gels of higher concentration (> 0.8%) require special attention when dissolving or re-melting. Make sure that the solution is completely clear of clumps or glassy granules. Distorted electrophoresis DNA band patterns will result if the gel is not properly prepared. 14. Electrophoretic separation: The tracking dye should travel at least 6 cm from the wells for adequate separation before staining. 15. Staining: Staining of higher concentration gels (> 0.8%) require additional care to obtain clear, visible results. After staining (15 to 30 min.) with InstaStain Ethidium Bromide or liquid ethidium bromide, examine the results using a UV (300 nm) transilluminator. Repeat the staining as required. Gels stained with InstaStain Blue or other liquid blue stain may fade with time. Re-stain the gel to visualize the DNA bands. 16. DNA 200 bp ladder: After staining the agarose gel, the DNA 200 bp ladder (markers) should be visible. If bands are visible in the markers and control lanes, but bands in the sample lanes are faint or absent, it is possible that DNA was not successfully extracted from the cells. If the ladder, control and DNA bands are all faint or absent, potential problems could include improper gel preparation, absence of buffer in the gel, improper gel staining or a dysfunctional electrophoresis unit or power source.