Mitochondrial DNA Analysis Using PCR

Transcription

1 The Biotechnology Education Company Revised and Updated Mitochondrial DNA Analysis Using PCR Storage: See Page 3 for specific storage instructions Experiment Objective: The objective of this experiment is for students to isolate human mitochondrial DNA and amplify two separate regions of the mitochondrial chromosome using the polymerase chain reaction. This experiment is designed for DNA staining with InstaStain Ethidium Bromide. EDVOTEK, Inc EDVOTEK

2 2 xxx Table of Contents Mitochondrial DNA Analysis Using PCR Experiment Components 3 Experiment Requirements 4 Background Information 5 Experiment Procedures Experiment Overview and General Instructions 10 Laboratory Safety 11 Module I: Isolation of DNA from Human Hair 12 Module II: Amplification of the Mitochondrial Regions 14 Module III: Separation of PCR Reactions by Agarose Gel Electrophoresis 16 Study Questions 18 Instructor's Guidelines Notes to the Instructor 20 Pre-Lab Preparations 23 Experiment Results and Analysis 25 Study Questions and Answers 26 Appendices A PCR Experimental Success Guidelines 28 B Polymerase Chain Reaction Using Three Waterbaths 30 C Preparation and Handling of PCR Samples With Wax 31 D 1.0% Agarose Gel Preparation 32 E 1.0% Agarose Gels - Quantity Preparations 33 F Staining and Visualization of DNA with InstaStain Ethidium Bromide Cards 34 G InstaStain Blue: One Step Staining and Destaining 35 Material Safety Data Sheets 36 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 Mitochondrial DNA Analysis Using PCR 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. Mitochondrial 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 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. UltraSpec-Agarose Electrophoresis Buffer (50x) 10x Gel Loading Solution InstaStain Ethidium Bromide InstaStain Blue Microcentrifuge Tubes PCR tubes (0.2 ml - for thermal cyclers with 0.2 ml template) Calibrated transfer pipets Wax beads (for waterbath option or thermal cyclers without heated lid) THIS EXPERIMENT DOES NOT CONTAIN HUMAN DNA. of the experiment components are derived from human sources. EDVOTEK - The Biotechnology Education Company EDVOTEK FAX: (301) info@edvotek.com

4 4 xxx Mitochondrial DNA Analysis Using PCR 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 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 Mitochondrial DNA Analysis Using PCR 5 Mitochondrial DNA Analysis Cristae Ribosomes DNA Figure 1: Structure of Mitochondrion Mitochondria (plural for mitochondrion) are the energy-producing organelles of the cell. Mitochondria are generally oblong or egg-shaped. Both plant and animal cells possess mitochondria. The number of mitochondria per cell varies depending on the cell type, ranging from only a few in skin cells to thousands in skeletal muscle cells. Outer membrane Matrix Inner membrane Unlike other organelles, mitochondria have two separate membranes. The outer membrane is fairly porous, possessing a protein called porin. The inner membrane, however, is highly impermeable to ions and is enriched in a rare, negatively charged phospholipid known as cardiolipin. The inner membrane is highly convoluted, with infoldings called cristae (Figure 1) that greatly increase the total membrane surface area. The inner membrane also contains the enzymes that catalyze cellular respiration, the process whereby energy is produced for the cell. Background Information Fat & Sugar Byproducts Citric Acid Cycle Fatty Acid Oxidation Electron Transport Chain Figure 2: Site of metabolic pathways that convert sugars and fats to energy. ATP Synthase ATP Energy for cell The space inside the inner membrane is known as the matrix (Figure 1). Within the matrix and inner membrane, the chemical reactions that produce energy for the cell take place. As shown in Figure 2, sugars and fatty acids, broken down to two carbon units, enter a series of reactions known as the citric acid or Krebs cycle. Sugars are broken down in the cytoplasm while fatty acids are broken down in the mitochondria by a process known as ß (beta) oxidation. The citric acid cycle generates electrons that enter the

6 6 Mitochondrial DNA Analysis Using PCR Mitochondrial DNA Analysis Background Information Complex I Genes Ribosomal RNA genes Complex IV Genes D-Loop 0/16569 PCR Products 8278 ATP Synthase Cytochrome B 9199 Figure 3: Genetic map of mitochondrial DNA Complex I Genes electron transport chain, a cluster of protein complexes that reside in the inner membrane of the mitochondria. In the final step of energy production, protons generated by the electron transport chain flow through a pump known as ATP synthase, driving the production of ATP, the primary energy-containing molecule used in biological systems. This final energy-producing process is known as oxidative phosphorylation. The DNA present in the matrix is distinct from the DNA found in the cell s nucleus. Mitochondrial DNA (mtdna) is contained in a single circular chromosome, shown in Figure 3, that has been completely sequenced. The mitochondrial chromosome contains 16,569 base pairs of DNA and 37 genes. MtDNA encodes 13 polypeptides, all of which are subunits of the respiratory chain complex. This is shown on the map in Figure 3, illustrating the I II III Figure 4: Patterns of inherited maternal mitochondrial diseases

7 Mitochondrial DNA Analysis Using PCR 7 Mitochondrial DNA Analysis locations of genes that encode proteins in complexes I and IV of the electron transport chain. As shown, mtdna also encodes mitochondrial ribosomal RNA and the ATP synthase, in addition to cytochrome B, another constituent of the electron transport chain. MtDNA encodes only part of the electron transport chain; nuclear DNA encodes the remaining complex subunits. One peculiarity is that mitochondrial protein synthesis uses a slightly different genetic code than cytoplasmic translation. As all cells possess only one nucleus but several hundred or thousand mitochondria, mtdna is present in great excess over nuclear DNA in most cells. This relative abundance of mtdna is taken advantage of by forensic investigators after obtaining crime scene specimens that are degraded or otherwise insufficient for nuclear DNA PCR analysis. The D-loop (Figure 3) has a high degree of variability between individuals and can be sequenced to demonstrate variations. MtDNA typing, however, cannot be used to conclusively link suspects to crime scenes; rather, it is used to include or exclude suspects for further scrutiny. During the past twenty years, an ever-increasing number of diseases have been shown to be due to mitochondrial dysfunction. These disorders result when mitochondrial ATP generation is insufficient to meet energy needs in a particular tissue. Because muscle and nerve cells contain large numbers of mitochondria, these organ systems are most affected by mitochondrial dysfunction. Mitochondrial diseases may be due to mutations in mtdna genes or mutations in nuclear genes that encode mitochondrial enzymes. Diseases caused by mtdna mutations include the myopathies, diseases that affect various muscles and encephalomyopathies, which cause both muscular and neurological problems. Huntington s chorea, a devastating disease that results in dementia and loss of motor control, is caused by defects in oxidative phosphorylation and has been mapped to a mutation in nuclear DNA encoding a non-mitochondrial protein. Other diseases such as Alzheimer s and Parkinson s disease involve mitochondrial abnormalities, although it is unclear how these abnormalities relate to disease pathology. Mitochondria also appear to play roles in aging and in programmed cell death, also known as apoptosis. Background Information Since mitochondria are present in the cytoplasm, they are inherited independently from the nucleus. A female egg cell possesses over 10,000 mitochondria, while a sperm cell has very few. Thus during fertilization, mitochondrial DNA is inherited almost exclusively from the mother. Although a small amount of paternal mtdna is present in the fertilized egg, this DNA appears to be selectively destroyed by the newly fertilized egg. This pattern of inheritance of mtdna is known as maternal inheritance. Maternal inheritance is indicated when all offspring, male and female, of the mother are afflicted with a specific condition (Figure 4). The severity of any particular mitochondrial disorder is highly variable, depending on the number of mutated mitochondria inherited from the mother (Figure 5). To examine mitochondrial DNA, the polymerase chain reaction (PCR) is usually employed. PCR, invented in 1984, has gained widespread use and its

8 8 Mitochondrial DNA Analysis Using PCR Mitochondrial DNA Analysis Background Information Mother with mild or no symptoms Mature oocyte Fertilization of oocyte number of mitochondria increases 80% mutant Child with severe disease 50% mutant Child with mild disease 20% mutant Figure 5: Mitochondrial inheritance nucleus mutant Child with no disease normal inventor, Kary Mullis, was awarded a Nobel Prize in The enormous utility of the PCR method is based on its ease of use and its ability to allow the amplification of small DNA fragments. The essence of the PCR method (Figure 6) is the use of an enzyme known as Taq polymerase. This enzyme, purified from a bacterium isolated from hot springs, is stable at very high temperatures. In the first step of PCR, known as denaturation, the DNA complementary strands are separated at 94 degrees, while the polymerase remains stable. In the second step, known as annealing, the sample is cooled to a temperature in the range of 42 C to 65 C. to allow hybridization of small (15-30 nucleotide) synthetic oligonucleotides, known as primers, to the target to be amplified. In the third step, known as extension, the temperature is raised to 72 C and the DNA polymerase then adds nucleotides to the primers to complete each new complementary strand of the target. These three steps constitute one cycle. This process is typically repeated for 25 to 50 cycles, amplifying the target exponentially (Figure 6). PCR is performed in an instrument known as a thermal cycler, which is programmed to heat the sample at the designated temperature for each step and then rapidly change temperatures for the following step. In this experiment, students will examine their mtdna from hair cells. To do this, PCR is used to amplify two separate regions of the mitochondrial chromosome, shown in Figure 3. Amplification of these regions will result in PCR products of 921 and 672 base pairs. Following PCR, the amplified DNA is then subjected to gel electrophoresis and staining for visualization.

9 Mitochondrial DNA Analysis Using PCR 9 Mitochondrial DNA Analysis Target Sequence = Separation of two DNA strands 3' 3' = Primer 1 Cycle 1 = Primer 2 3' 3' 3' 3' Denature 94 C Anneal 2 primers 40 C - 65 C The Experiment 3' 3' Extension 72 C Cycle 2 3' 3' 3' 3' 3' 3' Cycle 3 3' 3' 3' 3' 3' 3' 3' 3' Figure 6: Polymerase Chain Reaction

10 10 Mitochondrial DNA Analysis Using PCR Experiment Overview and General Instructions Before you start the experiment 1. Read all instructions before starting the experiment. 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 objective of this experiment is for students to isolate human mitochondrial DNA and amplify two separate regions of the mitochondrial chromosome using the polymerase chain reaction. Brief Description of Experiment: In this experiment, students examine their mtdna from hair. To do this, PCR is used to amplify two separate regions of the mitochondrial chromosome. Amplification of these regions will result in PCR products of 921 and 672 base pairs. Following PCR, the amplified DNA is then subjected to gel electrophoresis and staining for visualization. This experiment has three modules: I. Isolation of DNA from hair II. PCR Amplification of the Mitochondrial region 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 Gel concentration required 1.0%

11 Mitochondrial DNA Analysis Using PCR 11 Laboratory Safety 1. Gloves and goggles should be worn routinely as good laboratory practice. 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. The Experiment 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. Always wash hands thoroughly with soap and water after handling reagents or biological materials in the laboratory.

12 12 Mitochondrial DNA Analysis Using PCR Module I: Isolation of DNA from Human Hair 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. The Experiment 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 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.

13 Mitochondrial DNA Analysis Using PCR 13 Module I: Isolation of DNA from Human Hair, continued WARNING! Make sure you are using a screw-cap tube for boiling your sample. Do not use snap-top tubes when boiling. 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. 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. The Experiment 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 Mitochondrial regions. OPTIONAL STOPPING POINT The supernatant may be stored at -20 C until the experiment is continued.

14 14 Mitochondrial DNA Analysis Using PCR Module II: Amplification of the Mitochondrial Regions PCR Reaction: The Experiment 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. 1. Transfer the PCR Reaction pellet to the appropriate sized tube (e.g. 0.5 ml or 0.2 ml) for your thermal cycler. 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: Mitochondrial primer mix 20.0 µl Hair 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. 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: Mitochondrial primer mix 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. 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.

15 Mitochondrial DNA Analysis Using PCR 15 Module II: Amplification of the Mitochondrial Regions, continued 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 25 Final Extension 94 C for 4 min. 94 C for 1 min. 72 C for 5 min. 55 C for 1 min. 72 C for 2 min. 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. The Experiment 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.

16 16 Mitochondrial DNA Analysis Using PCR Module III: Separation of PCR Reactions by Agarose Gel Electrophoresis The Experiment 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. 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).

17 Mitochondrial DNA Analysis Using PCR 17 Module III: Separation of PCR Reactions by Agarose Gel Electrophoresis This experiment requires a 1.0% agarose gel and is designed for staining with InstaStain Ethidium Bromide. Loading DNA Samples 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 (optional) 3 Student #1 4 Student #2 5 Student #3 6 Student #4 The Experiment 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.

18 18 Mitochondrial DNA Analysis Using PCR Study Questions Answer the following study questions in your laboratory notebook or on a separate worksheet. 1. What are the three energy-producing sets of chemical reactions that take place inside the mitochondrion? The Experiment 2. How are mitochondria different from other organelles inside the cell? 3. Is it possible for a child to be healthy if his/her father is affected with a mitochondrial disease? From an unaffected mother? Why or why not? What might be some symptoms of such a disease? 4. If a crime scene sample is too degraded for normal DNA profiling, are any further analyses possible? If so, what assay(s) could be performed?

19 Mitochondrial DNA Analysis Using PCR 19 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

20 20 Mitochondrial DNA Analysis Using PCR 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 from hair II. PCR Amplification of the Mitochondrial region 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 (25 cycles) will take about minutes or can be processed overnight and held at 4 C. 3. 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 which follows. Time and Voltage 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.

21 Mitochondrial DNA Analysis Using PCR 21 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.

22 22 Mitochondrial DNA Analysis Using PCR 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 Ethidium Bromide (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.

23 Mitochondrial DNA Analysis Using PCR 23 Pre-Lab Preparations Module I: Isolation of DNA 1. Add 100 µl of 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. Suggestion: Cut the end of the pipet tip to avoid clogging of the pipet with beads in the chelating agent. 2. Prepare and dispense 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 into 13 tubes. Cap and mix the chelating agent in between each aliquot. Label these 13 tubes lysis solution. Each tube will be shared by a student pair. Instructor s Guide Each Student should receive: One 1.5 ml screw cap microtest tube One calibrated transfer pipet Reagents to be Shared by Two Students: 300 µl Lysis Solution 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.

24 24 Mitochondrial DNA Analysis Using PCR Pre-Lab Preparations Module II - Amplification of mitochondrial regions Instructor s Guide Each Student should receive: One tube containing a PCR reaction pellet One 0.5 ml microtest tube Reagents to be Shared by Two Students: 50 µl Mitochondrial Primer Mix 20 µl 10x Gel Load Solution 1. Thaw the mitochondrial primer mix (B) and place it on ice. Dispense 50 µl into 13 tubes. Label these 13 tubes Mito Primer. Distribute one tube for each pair of students 2. Dispense 20 µl of 10x Gel Loading Solution for each student pair. 3. There is material for one control PCR reaction. You may wish to perform the control reaction in your thermal cycler before the students try their reactions. 4. The Thermal cycler should be programmed for the following cycles. Initial Denaturation 25 Final Extension 94 C for 4 min. 94 C for 1 min. 72 C for 5 min. 55 C for 1 min. 72 C for 2 min. 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 1. 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. 2. Aliquot 30 µl of the 200 base-pair ladder (C) into microcentrifuge tubes. Distribute one tube of ladder per gel.

25 Mitochondrial DNA Analysis Using PCR 25 Experiment Results and Analysis Student #5 Student #4 Student #3 Student #2 Student #1 200 base pair ladder Student #5 Student #4 Student #3 Student #2 Student #1 200 base pair ladder 1200bp 1000bp 800bp 600bp 400bp 1200 bp 1000 bp 800 bp 600 bp 400 bp 200 bp Short gel Instructor s Guide 200bp Long Gel Idealized Schematics Students' PCR products should show two bands with lengths of 672 and 921 base pairs. Note: Depending on the PCR conditions used, a diffuse, small-molecular weight band, known as a "primer dimer", may be present below the 200 bp marker. 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

26 26 Mitochondrial DNA Analysis Using PCR Study Questions and Answers 1. What are the three energy-producing sets of chemical reactions that take place inside the mitochondrion? Instructor s Guide The three energy-producing reaction cascades that occur in the mitochondrion are: a) the citric acid, or Krebs, cycle b) fatty acid (beta) oxidation; and c) the electron transport chain (oxidative phosphorylation) 2. How are mitochondria different from other organelles inside the cell? Mitochondria have a double membrane and also have their own unique DNA chromosome, RNA, and ribosomes. 3. Is it possible for a child to be healthy if his/her father is affected with a mitochondrial disease? From an unaffected mother? Why or why not? What might be some symptoms of such a disease? Mitochondrial diseases are inherited maternally, exclusively from the mother. Therefore, a child cannot inherit a mitochondrial disease from an afflicted father. It is possible, however, for a child to be stricken with a mitochondrial disease inherited from a healthy mother who carries a small number of diseased mitochondria, if these defective mitochondria are incorporated into the oocyte that becomes fertilized. Mitochondrial disorders primarily affect the muscular and nervous systems, as these organs possess large numbers of mitochondria. Typical symptoms of muscle disorders include muscle wasting and exercise intolerance. Nervous system disorders can result in headaches, seizures, and hearing and visual impairment. 4. If a crime scene sample is too degraded for normal DNA profiling, are any further analyses possible? If so, what assay(s) could be performed? If a crime scene specimen is too degraded for standard DNA fingerprinting (which is performed on nuclear DNA) it may still be possible to analyze the mitochondrial DNA. This further analysis is possible because most cells typically possess many copies of mitochondrial DNA but only one nuclear DNA copy.

27 Mitochondrial DNA Analysis Using PCR 27 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

28 28 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. HUMAN HAIR Shaft Sheath Root 2. 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. 3. 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). 4. 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. 5. 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. 6. 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.

29 Appendix A PCR Experimental Success Guidelines (continued) 29 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: Higher concentration gels (> 0.8%) may require additional staining 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.

30 30 Appendix B Polymerase Chain Reaction Using Three Waterbaths Superior PCR results are obtained using an automated thermal cycler. However, if you do not have a thermal cycler, this experiment can be adapted to use three waterbaths (Cat. # 544). Much more care needs to be taken when using the three-waterbath PCR method. The PCR incubation sample is small and can easily be evaporated. Results using three waterbaths are often variable. Please refer to the Appendix entitled "PCR Samples with Wax Overlays" for sample handling and preparation tips. Each PCR Reaction pellet contains Taq DNA polymerase, four deoxytriphosphates, Mg +2 and buffer. Preparation of the PCR Reaction: 1. The PCR reaction sample should be prepared as specified in the experiment instructions. Each PCR reaction sample contains three critical components: PCR Reaction pellet Primer mix DNA for amplification 2. After adding the components of the PCR reaction sample, use clean forceps to transfer one wax bead to the PCR tube. At the start of the PCR reaction, the wax will melt and overlay the samples to prevent evaporation during heating. POLYMERASE chain reaction CYCLING 3. In the three-waterbath PCR method, the PCR reaction sample is sequentially cycled between three separate waterbaths, each set at different temperatures, for a specified period of time. The sequential placement of the reaction sample in the waterbaths maintained at three different temperatures constitutes one PCR cycle. One example of a PCR cycle might be as follows: Important Note It is imperative that the temperatures are accurately maintained throughout the experiment. 94 C for 1 minute 50 C for 1 minute 72 C for 1 minute See experiment instructions for specific program requirements. 4. The PCR tube must be handled carefully when sequentially cycled between the three waterbaths. For each cycle: Carefully place the PCR tube in a waterbath float. Make sure that the sample volume is at the bottom of the tube and remains undisturbed. If necessary, pulse spin the tube in a balanced microcentrifuge, or shake the tube to get all of the sample to the bottom of the tube. Use forceps to carefully lower the waterbath float (with tubes) sequentially into the waterbaths. 5. Process the PCR reaction sample for the total number of cycles specified in the experiment instructions. On the final cycle the 72 C incubation can be extended to 5 minutes. 6. After all the cycles are completed, the PCR sample is prepared for electrophoresis.

31 Appendix C Preparation and Handling of PCR Samples With Wax For Thermal Cyclers without Heated Lids, or PCR Using Three Waterbaths 31 Automated thermal cyclers with heated lids are designed to surround the entire sample tube at the appropriate temperature during PCR cycles. Heating the top of the tubes during these cycles prevents the very small sample volumes from evaporating. For thermal cyclers without heated lids, or when conducting PCR by the three-waterbath method, it is necessary to add a wax bead to the reaction sample. During the PCR process, the wax will melt and overlay the samples to prevent evaporation during heating. Each PCR Reaction pellet contains Taq DNA polymerase, four deoxytriphosphates, Mg +2 and buffer. preparing the PCR Reaction: 1. The PCR reaction sample should be prepared as specified in the experiment instructions. Each PCR reaction sample contains the following three critical components: PCR Reaction pellet Primer mix DNA for amplification 2. After adding the components of the PCR reaction sample, use clean forceps to transfer one wax bead to the PCR tube. 3. Process the PCR reaction sample for the total number of cycles specified in the experiment instructions. Preparing the PCR Reaction for electrophporesis: 4. After the cycles are completed, transfer the PCR tube to a rack and prepare the PCR sample for electrophoresis. Place the PCR tube in a 94 C waterbath long enough to melt the wax overlay. Use a clean pipet to remove most of the melted wax overlay. Allow a thin layer of the wax to solidify. Use a clean pipet tip to gently poke a hole through the solidified wax. Remove the tip. Use another clean pipet tip to enter the hole to remove the volume of mixture specified in the experiment instructions. Transfer this volume to a clean tube. Add other reagents according to experiment instructions, if applicable,. Add 5 µl of 10x Gel Loading solution to the sample and store on ice. 5. Proceed to delivery of the sample onto an agarose gel for electrophoresis as specified in the experiment instructions.