In Search of The Alcohol Gene

Transcription

1 The Biotechnology Education Company In Search of The Alcohol Gene Storage: See Page 3 for specific storage instructions EXPERIMENT OBJECTIVE: The objectives of this experiment are to amplify a specific region of human DNA representing a polymorphic region within the ADH3 gene and to analyze for the DNA polymorphisms by restriction enzyme analysis to determine the genetic basis for differences in the metabolism of alcohol. EDVOTEK, Inc EDVOTEK EVT

2 2 xxx In Search of the Alcohol Gene Table of Contents Experiment Components 3 Experiment Requirements 4 Background Information 5 Experiment Procedures Experiment Overview and General Instructions 10 Laboratory Safety 11 Module I: The Polymorphic Region withing the ADH3 Gene 12 Module II: Restriction Enzyme Analysis of PCR Reactions 13 Module III: Electrophoresis of Restriction Enzyme Digestion 13 Study Questions 17 Instructor's Guidelines Notes to the Instructor 19 Pre-Lab Preparations 22 Experiment Results and Analysis 24 Study Questions and Answers 25 Appendices A PCR Experimental Success Guidelines 27 B Polymerase Chain Reaction Using Three Waterbaths 28 C Preparation and Handling of PCR Samples With Wax 29 D 1.0% Agarose Gel Preparation 30 E 1.0% Agarose Gels - Quantity Preparations 31 F Staining and Visualization of DNA with InstaStain Ethidium Bromide Cards 32 Material Safety Data Sheets 33 The PCR process and Taq DNA polymerase are covered by patents owned by Hoffman-LaRoche, Inc. EDVOTEK -

3 In Search of the Alcohol Gene 3 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 suffi cient volume for pipeting. Spin samples for seconds at maximum speed. Experiment Components 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 PCR Primer mix -20 C Freezer C 200 base pair ladder -20 C Freezer D Control Polymorphic (heterozygous) -20 C Freezer E Ultrapure Water (dh 2 0) -20 C Freezer F-1 Template DNA (unknown 1) -20 C Freezer F-2 Template DNA (unknown 2) -20 C Freezer F-3 Template DNA (unknown 3) -20 C Freezer G Dryzyme Eco RI Refrigerator H Restriction Enzyme Reaction Buffer Refrigerator I Dryzyme Reconstitution Buffer -20 C Freezer 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 powder Electrophoresis Buffer (50x) 10x Gel Loading Solution InstaStain Ethidium Bromide Microcentrifuge Tubes (1.5 ml screw-cap tube use for boiling) PCR tubes (0.2 ml - for thermal cyclers with 0.2 ml template) Wax beads (for waterbath option or thermal cyclers without heated lid) THIS EXPERIMENT DOES NOT CONTAIN HUMAN DNA. None of the experiment components are derived from human sources. EDVOTEK - The Biotechnology Education Company EDVOTEK FAX: info@edvotek.com EVT

4 4 xxx In Search of the Alcohol Gene Experiment 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 signifi cantly higher rate of success. Thermal cycler (EDVOTEK Cat. # 541 highly recommended) or three waterbaths* Horizontal gel electrophoresis apparatus D.C. power supply Balance Microcentrifuge 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: Web: info@edvotek.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 In Search of the Alcohol Gene 5 Alcohol Metabolism ALCOHOL METABOLISM AND ITS EFFECTS The most socially accepted addictive drug, alcohol (ethanol, ethyl alcohol), presents life-threatening hazards because it acts as a central nervous system depressant that results in a reduction of the brain s functional activity. At low doses, it can have a stimulating effect that is manifested as excitement, euphoria, exhilaration and poor coordination. As alcohol doses increase, there is a progressive depression of brain function that leads to drowsiness, slurred speech, staggering and sedation. At higher levels of blood alcohol, nausea and vomiting and even death can occur. The amount of alcohol needed to produce its effects depends upon variables such as the individual s age, weight, sex, physical condition, co-ingestion of food, level of tolerance and genetic makeup. The level of alcohol intoxication can be measured by quantifying the amount of alcohol present in the blood stream using either a breathalyzer or blood test. This measurement, known as Blood Alcohol Concentration (BAC), corresponds to the number of grams of alcohol per deciliter (100 ml) of blood. A BAC of 0.10 usually results in impaired coordination and slurred speech; 0.50 can cause coma, and higher amounts can cause respiratory failure and death. Because of the known association of alcohol intoxication with car accidents, the legal limit of the BAC measure for automobile driving in most states is Background Information Alcohol is absorbed into the blood from both the stomach (20%) and intestine (80%), and is then distributed throughout the body in body fluids, that include blood, urine, saliva, spinal fluid, and tears and has the ability to damage or destroy various organs of the human body. Fortunately, humans metabolize blood alcohol in a two-step process that mainly occurs in the liver. The first metabolic step enzymatically converts alcohol to acetaldehyde, a compound that is more toxic than alcohol and is a major cause of hangovers. The enzyme involved in this reaction is called alcohol dehydrogenase (ADH). Acetaldehyde is then converted in the liver by aldehyde dehydrogenase (ALDH), to acetic acid. The end products of acetic acid in alcohol metabolism are carbon dioxide and water that are eliminated through the kidneys and lungs. NAD NADH + H + NAD + NADH + H + Ethanol Acetaldehyde Acetic acid ADH ALDH EDVOTEK, Inc. Copyright 2006, 2011, 2012 EDVOTEK, Inc., all rights reserved EVT

6 6 In Search of the Alcohol Gene Alcohol Metabolism, continued IDENTIFYING THE POLYMORPHISMS RELATED TO ALCOHOL METABOLISM Background Information Genetic research holds great promise for furthering the understanding of human responses to alcohol, with an emphasis on how alcohol is metabolized and causes of its addictive tendencies. Studies have shown that the human ability to metabolize alcohol and behavioral responses to alcohol are partially dependent upon the genetic makeup of individuals. Although humans are 99.9% genetically identical, research has revealed genetic variations among individuals that involve single DNA base pair changes called polymorphisms. Polymorphisms contribute to differences among individuals by altering the primary structure of a protein or enzymatic activity. It should be stressed that gene variants caused by polymorphisms can be responsible for predisposition to alcoholism and personality traits that can influence the likelihood of developing addictive behavior and the severity of secondary diseases resulting from chronic alcohol uptake. Human metabolism of alcohol can involve the participation of multiple forms of alcohol dehydrogenase and aldehyde dehydrogenase. Interestingly, humans are known to harbor different polymorphisms in the genes associated with the enzymatic activity. These modified gene forms that have dramatic effects on how alcohol is metabolized. For example, two polymorphisms in the ADH3 gene, ADH3*1 and ADH3*2, result in proteins with different enzymatic activities. The ADH3*1 is associated with the fast rate of ethanol oxidation that leads to high levels of acetaldehyde resulting accumulation of this toxic compound in the bloodstream. A build-up of toxic acetaldehyde in blood makes people who consume alcohol uncomfortable and ill. Therefore, individuals carrying the ADH3*1 are encouraged to consume modest amounts of alcohol to avoid build up of acetaldehyde in blood. The recent advances in human genetics has made possible to isolate human genes and identify polymorphisms in DNA. Such differences in genes that encode for proteins that are involved in alcohol metabolism can be detected and analyzed. Analysis utilize the polymerase chain reaction (PCR) reaction followed by restriction enzyme digestion of is one such combination that can be applied. THE POLYMERASE CHAIN REACTION (PCR) The (PCR) reaction is a DNA amplification technique that revolutionized almost all aspects of biological research. The procedure was invented in 1984 by Dr. Kary Mullis while at Cetus Corporation. Mulis was awarded a Nobel Prize for his work in PCR amplification can produce millions of copies from a small quantity of DNA. The enormous utility of PCR is based on its procedural simplicity and specificity. Since the first application of PCR to diagnose sickle cell anemia, a large number of amplifications have successfully been developed. PCR has made amplification of DNA an effective alternative to cloning. It is currently routinely used in forensics, paternity/kinship testing, and the identification of human remains. In preparation for PCR amplification, a set of DNA primers is designed to specifically amplify or produce copies of a small region of the genomic sequence containing the gene EDVOTEK, Inc. Copyright 2006, 2011, 2012 EDVOTEK, Inc., all rights reserved EVT

7 In Search of the Alcohol Gene 7 Alcohol Metabolism, continued of interest. The primers are two synthetic oligonucleotides typically base pairs in length that are synthesized so that they correspond to the start and end of a specific region of the DNA sequence to be amplified. The region of DNA selected to be amplified is known as the target. When identifying polymorphisms related to alcohol metabolism, DNA from individuals who show differences in this metabolic process is extracted for PCR amplification.. The extracted DNA is called the template. Freshly isolated DNA from biological sources will yield the best amplification. DNA extracted from stored specimens may be degraded and therefore less suitable for amplification. In addition to the two primers, the four deoxynucleotides (datp, dctp, dgtp, and dttp) that are the precursors building blocks of DNA and a thermally stable DNA polymerase are required. The most commonly used DNA polymerase is the enzyme Taq polymerase, which is purified from a bacterium that inhabits hot springs. This enzyme is stable at near-boiling temperatures. The PCR process requires a sequential heating and cooling cycle of the mixture at three different temperatures. It is efficiently performed in a thermal cycler, an instrument that is programmed to rapidly heat, cool, and maintain samples at designated temperatures for varying amounts of time. In the first step of the PCR reaction the mixture is heated to near boiling (94 C) to allow the two complementary DNA strands to be denatured. This step, known as denaturation, disrupts the hydrogen bonds between the strands and causes the complete separation of the two DNA strands. In the second PCR step, the sample is cooled to a temperature in the range of C. In this step, known as annealing, the two primers, which are present in great excess to the separated DNA template strands, bind to their target complements. In the third step, known as extension (also called DNA synthesis), the temperature is raised to an intermediate value (usually 72 C). At this temperature the Taq polymerase is maximally functional. It adds the precursor nucleotides to the primers to complete the synthesis of the new complementary strands. Background Information These three steps--denaturation, annealing, and extension constitute one PCR cycle. Each cycle doubles the amount of the target DNA. Calculated mathematically, if the cycle is repeated n times the number of copies will be an exponential enlargement of 2n. For example, ten cycles will produce 210 or 1,048,576 copies. The PCR process is typically repeated for cycles, theoretically amplifying the target sequence to millions of copies. In practice, however, the amount of product reaches a maximum after about 35 cycles. This is due to the depletion of reaction components and loss of DNA polymerase activity. The exact temperature and incubation time required for each of the three steps depends upon several factors, including the length of the DNA target and the Guanine /Cytosine (GC) content of the primer/target. One common problem that occurs during PCR is the production of unwanted amplification products. This may be due to contamination of the sample or non-specific annealing (to the wrong segment). If this were to occur in an early cycle, the incorrect copy will also be amplified. To reduce contamination, autoclaved tubes and pipet tips, as well as sterile water should be used. Gloves should always be worn when performing PCR. Minimizing the concentration of the primers may curtail the production of unwanted PCR due to nonspecific annealing. Another common technique is a hot start step, in which the PCR reagents are introduced in the reaction only after the DNA is fully denatured at 94 C. EDVOTEK, Inc. Copyright 2006, 2011, 2012 EDVOTEK, Inc., all rights reserved EVT

8 8 In Search of the Alcohol Gene Target Sequence = Separation of two DNA strands 3' 3' Background Information Cycle 1 = = Primer 1 Primer 2 3' 3' 3' 3' Denature 94 C Anneal 2 primers 40 C - 65 C 3' 3' Extension 72 C Cycle 2 3' 3' 3' 3' 3' 3' Cycle 3 3' 3' 3' 3' 3' 3' 3' 3' Figure 1: Polymerase Chain Reaction EDVOTEK, Inc. Copyright 2006, 2011, 2012 EDVOTEK, Inc., all rights reserved EVT

9 In Search of the Alcohol Gene 9 Alcohol Metabolism, continued RESTRICTION ENZYME DIGESTION Restriction enzymes are endonucleases which catalyze the cleavage of the phosphate bonds within both strands of DNA. The distinguishing feature of restriction enzymes is that they only cut at very specific palindromic sequences of bases. A single base change in the sequence results in the inability of the restriction enzyme to cut the DNA at that location. Differences in the sequence of DNA at a specific location can be quickly identified using restriction enzyme digestion. Restriction enzymes are named according to the organism from which they are isolated. This is done by using the first letter of the genus followed by the first two letters of the species. Finally, a Roman numeral is always used to designate one out of possibly several different restriction enzymes produced by the same organism or by different substrains of the same strain. A restriction enzyme requires a specific double-stranded recognition sequence of nucleotide bases to cut DNA. Recognition sites are usually 4 to 8 base pairs in length. Cleavage occurs within or near the site. The cleavage positions are indicated by arrows. Recognition sites are frequently symmetrical, i.e., both DNA strands in the site have the same base sequence when read 5 to 3. Such sequences are called palindromes. Consider the recognition site and cleavage pattern of Eco RI as an example. Background Information 5 -GAATTC-3 Eco RI 5 -G AATTC-3 3 -CTTAAG-5 Digestion 3-CTTAA G-5 The presence and absence of the restriction site can be treated as two alleles. The two alleles of a gene are a polymorphisim for that gene. In the example of the Alcohol Dehydrogenase gene (ADH) one allele has an EcoRI restriction enzyme recognition site in the middle of the region and is digested. The second allele has a single base change in the recognition site and the DNA is not digested by the enzyme. PCR-RFLP ANALYSIS OF POLYMORPHISM To identify polymorphisms in the DNA scientists use RFLP analysis (Restriction Fragment Length Polymorphism). When two of more samples of DNA from different individuals containing an identical region of a chromosome are compared by digestion with the same restriction enzyme, polymorphic changes can be visualized by differences in the restriction fragment lengths. If all the strands of DNA were identical they would have identical restriction fragment patterns. Any changes in the restriction enzyme digestion pattern is due to changes in the DNA sequence that adds or removes restriction enzyme digestion sites on the DNA and can be seen as changes in the resulting fragment lengths. By using PCR scientists can amplify a specific region of the DNA quickly and test that DNA fragment for digestion. When the amplification primers are designed to amplify only the region of DNA if interest the test for the presence of the polymorphism can be quickly accomplished for a large number of samples. In this experiment the students will use the PCR-RFLP method to test several samples for the presence of polymorphisms. EDVOTEK, Inc. Copyright 2006, 2011, 2012 EDVOTEK, Inc., all rights reserved EVT

10 10 In Search of the Alcohol Gene 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 objectives of this experiment are to amplify a specific region of human DNA representing a polymorphic region within the ADH3 gene and to analyze for the DNA polymorphisms by restriction enzyme analysis to determine the genetic basis for differences in the metabolism of alcohol. BRIEF DESCRIPTION OF EXPERIMENT: In this experiment, each group of students will amplify a specific region of simulated human DNA by PCR. Polymorphic sites within the ADH3 gene from two samples will be analyzed by restriction enzyme analysis to determine the genetic basis for the differences in the metabolism of alcohol. GEL SPECIFICATIONS This experiment requires a gel with the following specifications: Recommended gel size 7 x 7 cm (short tray) Number of sample wells required 6 Placement of well-former template first set of notches Gel concentration required 1.0% EDVOTEK, Inc. Copyright 2006, 2011, 2012 EDVOTEK, Inc., all rights reserved EVT

11 In Search of the Alcohol Gene 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. Turn off power and then unplug the equipment when not in use. Electrical current from the power source is automatically disrupted when the electrophoresis cover is removed from the apparatus on all EDVOTEK models. However, exercise caution when working with electrophoresis equipment. After electrophoresis is completed, turn off the power, then unplug the power source before disconnecting the leads and removing the cover. The Experiment 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. EDVOTEK, Inc. Copyright 2006, 2011, 2012 EDVOTEK, Inc., all rights reserved EVT

12 12 In Search of the Alcohol Gene Module I: The Polymorphic Region within the ADH3 Gene PCR REACTION: The Experiment 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 3 tubes containing PCR Reaction Pellet 1, 2, and 3. Put your initials on the tubes. 3. Tap the reaction tube to assure the reaction pellet is at the bottom of the tube. 4. Prepare the following PCR reaction tubes as summarized in Chart 1, below. Use a FRESH micropipet tip for each transfer of sample. Chart 1 - Summary of PCR reaction tubes Tube 1 Unknown 1 Tube 2 Unknown 2 Tube 3 Unknown 3 PCR reaction pellets TM 1 pellet 1 pellet 1 pellet PCR Primer mix (C) 5 μl 5 μl 5 μl DNA Sample (F-1; F-2; or F-3) 5 μl 5 μl 5 μl Ultrapure H 2 0 (E) 15 μl 15 μl 15 μl Final volume 25 μl 25 μl 25 μl 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 suffi cient volume for pipeting. Spin samples for seconds at maximum speed. 5. Gently mix the PCR reaction tubes and quick spin them in a microcentrifuge to collect all he sample at the bottom of the tube. Make sure reagents are 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. POLYMERASE CHAIN REACTION CYCLING 7. For automatic cycling, each student should place his/her PCR tubes in the programmed thermal cycler. Follow the same cycling schedule if you are manually cycling in three water baths. Initial Denaturation 94 C for 180 sec. 30 Cycles 94 C for 45 sec. 55 C for 45 sec. 72 C for 45 sec. Final Extension 72 C for 180 sec. OPTIONAL STOPPING POINT After the 30 cycles are completed, the samples can be held in the thermal cycler at 4 C or frozen until ready for restriction enzyme analysis. EDVOTEK, Inc. Copyright 2006, 2011, 2012 EDVOTEK, Inc., all rights reserved EVT

13 In Search of the Alcohol Gene 13 Module II: Restriction Enzyme Digestion of Amplified PCR Reactions RESTRICTION ENZYME DIGESTION OF PCR PRODUCTS TO DETECT DIFFERENT POLYMORPHISMS 1. Remove the PCR reactions from the thermal cycler (after completing 30 cycles) or from storage in the freezer. 2. Label 3 new tubes for the Restriction Enzyme Digests: Tube 1, Tube 2, and Tube 3. Put your initials on the tubes. 3. Prepare the following restriction enzyme digest reaction mixtures as summarized in chart 2. Use a FRESH micropipet tip for each transfer of sample. Chart 2 Summary of restriction enzyme digestion tubes PCR product Tube 1 (F-1) PCR product Tube 2 (F-2) PCR product Tube 3 (F-3) DNA sample from the 5 μl 5 μl 5 μl PCR reaction tube Restriction Enzyme Reaction Buffer (H) 5 μl 5 μl 5 μl Ultrapure H 2 0 (E) 5 μl 5 μl 5 μl The Experiment Rehydrated dryzyme (G) 5 μl 5 μl 5 μl Final volume 20 μl 20 μl 20 μl 4. Cap the reaction tubes and tap gently to mix. Then tap each tube on the lab bench to collect contents at the bottom. 5. Incubate the tubes for 1 hour in the 37 C waterbath. 6. Prepare the remaining sample in the PCR reaction tubes (tubes 1, 2, and 3) for electrophoresis by adding 5 µl of 10x gel loading solution to each tube. Set these tubes aside. 7. After the incubation of the restriction enzyme reactions, add 5 µl of 10x gel loading solution to each of the Eco RI digestion tubes (Tube 1, Tube 2, and Tube 3). Module III: Electrophoresis of Restriction Enzyme Digestion AGAROSE GEL REQUIREMENTS Recommended gel size: 7 x 7 cm (short tray) Number of sample wells required: 6 sample wells Placement of well-former template: first set of notches Agarose gel concentration: 1.0% EDVOTEK, Inc. Copyright 2006, 2011, 2012 EDVOTEK, Inc., all rights reserved EVT

14 14 In Search of the Alcohol Gene Module III: Electrophoresis of Restriction Enzyme Digestion AGAROSE GEL REQUIREMENTS This experiment requires a 1.0% agarose gel. The Experiment Wear gloves and safety goggles If using EDVOTEK electrophoresis units, cast a gel in the 7 x 7 cm gel casting tray. Use an 6-well comb to cast the required number wells for this experiment. 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. 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. Important Note Continue heating until the fi nal solution appears clear (like water) without any undissolved particles. Check the solution carefully. If you see "crystal" particles, the agarose is not completely dissolved. 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). This experiment requires a 1.0% agarose gel and is designed for staining with InstaStain Ethidium Bromide. EDVOTEK, Inc. Copyright 2006, 2011, 2012 EDVOTEK, Inc., all rights reserved EVT

15 In Search of the Alcohol Gene 15 Module III: Electrophoresis of Restriction Enzyme Digestion LOADING DNA SAMPLES This experiment requires two 1.0% agarose gels per group. Gels can be run in sequence or simultaneously depending upon equipment availability. Reminder: Before loading the samples, make sure the gel is properly oriented in the apparatus chamber. Black Sample wells + Red Have a water bath or beaker of water warmed to 50 C for heating tubes containing DNA ladder fragments before gel loading. 1. Heat the 200 bp DNA ladder (C), Control Polymorphic DNA (D) 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 of the samples in the following sequence: Lane bp DNA ladder (C) 2 Control Polymorphic DNA (D) 3 Unknown 1 - undigested PCR Product F-1 4 Unknown 2 - undigested PCR Product F-2 5 Unknown 3 - undigested PCR Product F-3 The Experiment 3. Load Samples - Gel 2: Load entire volume of the samples in the following sequence: Lane bp DNA ladder 2 Control Polymorphic DNA 3 Unknown 1 - Eco RI digest PCR Product F-1 4 Unknown 2 - Eco RI digest PCR Product F-2 5 Unknown 3 - Eco RI digest PCR Product F-3 3. Record the position of your sample in the gel for easy identification after staining. EDVOTEK, Inc. Copyright 2006, 2011, 2012 EDVOTEK, Inc., all rights reserved EVT

16 16 In Search of the Alcohol Gene Module III: Electrophoresis of Restriction Enzyme Digestion RUNNING THE GEL 1. After the DNA samples are loaded, properly orient the cover and carefully snap it onto the electrode terminals. The Experiment 2. Insert the plugs of the black and red leads into the corresponding inputs of the power source. 3. Set the power source at the required voltage and conduct electrophoresis for the length of time determined by your instructor. General guidelines are presented in Table C in the instruction Appendix provided by your instructor. 4. Check to see that current is flowing properly - you should see bubbles forming on the two platinum electrodes. 5. 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. EDVOTEK, Inc. Copyright 2006, 2011, 2012 EDVOTEK, Inc., all rights reserved EVT

17 In Search of the Alcohol Gene 17 Study Questions Answer the following study questions in your laboratory notebook or on a separate worksheet. 1. What are variables that contribute to alcohol intoxication? 2. How is alcohol absorbed and distributed upon ingestion? 3. What are polymorphisms in DNA and how do they contribute to differences among individuals? 4. How is PCR used to determine human genetics and identify polymorphisms in DNA? 5. What are the three steps in a PCR cycle and what does each step accomplish? 6. What are the enzymatic steps involved in alcohol metabolism? The Experiment EDVOTEK, Inc. Copyright 2006, 2011, 2012 EDVOTEK, Inc., all rights reserved EVT

18 18 xxx In Search of the Alcohol Gene Notes: EDVOTEK -

19 In Search of the Alcohol Gene 19 Instructor s Guide OVERVIEW OF LABORATORY INVESTIGATIONS The hands-on laboratory experience is a very important component of the science courses. Laboratory experiment activities allow students to identify assumptions, use critical and logical thinking, and consider alternative explanations, as well as help apply themes and concepts to biological processes. EDVOTEK experiments have been designed to provide students the opportunity to learn very important concepts and techniques used by scientists in laboratories conducting biotechnology research. Some of the experimental procedures may have been modified or adapted to minimize equipment requirements and to emphasize safety in the classroom, but do not compromise the educational experience for the student. The experiments have been tested repeatedly to maximize a successful transition from the laboratory to the classroom setting. Furthermore, the experiments allow teachers and students the flexibility to further modify and adapt procedures for laboratory extensions or alternative inquiry-based investigations. 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: Web: info@edvotek.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 ORGANIZING & IMPLEMENTING THE EXPERIMENT 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 ( ). 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: info@edvotek.com EVT

20 20 In Search of the Alcohol Gene Notes to the Instructor: APPROXIMATE TIME REQUIREMENTS 1. The PCR step will take approximately 70 minutes respectively if using a thermal cycler. The option which uses three waterbaths will take longer. The Experiment 2. Agarose gel preparation: Your schedule will determine when to prepare the gel(s) for an experiment. Whether you choose to prepare the gel(s) or have the students do it, allow approximately minutes for this procedure. Generally, 20 minutes of this time is required for gel solidification. 3. The approximate time for electrophoresis will vary from 30 minutes to 2 hours or more, depending on the power supply voltage. Generally, the higher the voltage applied the faster the samples migrate. However, the maximum amount of voltage significantly depends upon the design of the electrophoresis apparatus and should not exceed manufacturers recommendations. Time and Voltage recommendations for EDVOTEK equipment are outlined in Table C. NOTE: Gel 1 (PCR product gel) can be run and stained while the restriction enzyme digestion of PCR products incubates. Table C Time and Voltage (1.0% - 7 x 7 cm gel) Volts Recommended Time Minimum Maximum min 45 min 90 min 45 min 60 min 120 min EDVOTEK, Inc. Copyright 2006, 2011, 2012 EDVOTEK, Inc., all rights reserved EVT

21 In Search of the Alcohol Gene 21 Notes to the Instructor: STAINING AND VISUALIZATION OF PCR PRODUCTS AFTER AGAROSE GEL ELECTROPHORESIS For this experiment, optimal visualization of PCR products will be obtained by staining gels with InstaStain Ethidium Bromide (InstaStain EtBr) cards, which are included in this experiment kit. 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. LABORATORY NOTEBOOKS It is highly recommended that students maintain a laboratory notebook to formulate hypotheses and to record experimental procedures and results. Instructor s Guide EDVOTEK, Inc. Copyright 2006, 2011, 2012 EDVOTEK, Inc., all rights reserved EVT

22 22 In Search of the Alcohol Gene Pre-Lab Preparations MODULE I: Amplification of the Polymorphic Region Within the ADH3 Gene Instructor s Guide 1. Program the thermal cycler for the following setup for 30 cycles. Follow the same cycling schedule if you are manually cycling in three water baths. Initial Denaturation 30 Cycles Final Extension 94 C for 180 sec. 94 C for 45 sec. 72 C for 180 sec. 55 C for 45 sec. 72 C for 45 sec. 2. Place components primer solutions and DNAs on ice. Aliquot the indicated amounts to each tube using a fresh pipet tip for each component. Alternatively, you can set up a pipeting station for the class. Each Group (4 students) Requires: One tube of each of the following (3 Reactions per group): F-1 DNA (Unknown 1) 8 μl F-2 DNA (Unknown 2) 8 μl F-3 DNA (Unknown 3) 8 μl Primer Solution (B) 25 μl Ultrapure Water (E) 100 μl Tubes with PCR reaction pellets 3 tubes Wax beads (use only if required) 6 Notes and Reminders: Accurate temperatures and cycle times are critical. A pre-run for one cycle (approx. 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. 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. EDVOTEK, Inc. Copyright 2006, 2011, 2012 EDVOTEK, Inc., all rights reserved EVT

23 In Search of the Alcohol Gene 23 Pre-Lab Preparations MODULE II: RESTRICTION ENZYME ANALYSIS AND ELECTROPHORESIS Rehydrate Dryzyme 1. Add 85 µl of Dryzyme Reconstitution Buffer (I) to the tube and set on ice 1 minute. 2. Mix the tube for 30 seconds (vortex or tap bottom of the tube). 3. Add 85 µl Ultrapure Water (E). 4. Mix the tube for 30 seconds (vortex or tap bottom of the tube). 5. Set the tube on ice. Instructor s Guide Restriction Enzyme Digestion For each group, aliquot the following: Rehydrated Dryzyme (G) 16 µl Restriction Enzyme Reaction Buffer (H) 20 µl 10x Gel Loading Solution 50 µl MODULE III: Electrophoresis of Restriction Enzyme Digestion For each group, aliquot the following: 200 bp Ladder (C) 50 µl Control Polymorphic DNA Digest (heterozygous) (D) 50 µl EDVOTEK, Inc. Copyright 2006, 2011, 2012 EDVOTEK, Inc., all rights reserved EVT

24 24 In Search of the Alcohol Gene Experiment Results and Analysis PHOTOS OF GEL RESULTS Instructor s Guide Gel 1: PCR Reaction for Alcohol Dehydrogenease Lane bp DNA ladder (C) 2 Control Polymorphic DNA Digest (heterozygous) (D) 3 Unknown 1 undigested PCR Product F-1 4 Unknown 2 undigested PCR Product F-2 5 Unknown 3 undigested PCR Product F-3 Gel 2: RFLP Analysis to Detect Alcohol Dehydrogenase Polymorphisms Lane bp DNA ladder (C) 2 Control Polymorphic Heterozygous DNA (D) 3 Unknown 1 - Eco RI digest of PCR Product F-1 4 Unknown 2 - Eco RI digest of PCR Product F-2 5 Unknown 3 - Eco RI digest of PCR Product F-3 EDVOTEK, Inc. Copyright 2006, 2011, 2012 EDVOTEK, Inc., all rights reserved EVT

25 Please refer to the kit insert for the Answers to Study Questions

26 26 xxx In Search of the Alcohol Gene Appendices A B C D E F 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 Material Safety Data Sheets EDVOTEK -

27 Appendix A PCR Experimental Success Guidelines 27 The following guidelines offer some important suggestions, reminders and hints for maximizing success. THE PCR REACTION 1. Add Primers and DNA to the PCR Reaction Bead: Add the primer mixture (forward and reverse primers) and the template DNA 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. 2. 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. 3. 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. 4. 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 5. 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. 6. Electrophoretic separation: The tracking dye should travel at least 4.5 cm from the wells for adequate separation before staining. 7. 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. 8. DNA 200 bp ladder: After staining the agarose gel, the DNA 200 bp ladder (markers) should be visible. If the ladder 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. EDVOTEK, Inc. Copyright 2006, 2011, 2012 EDVOTEK, Inc., all rights reserved EVT

28 28 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 And water (optional) 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 Important Note It is imperative that the temperatures are accurately maintained throughout the experiment. 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: 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. EDVOTEK, Inc. Copyright 2006, 2011, 2012 EDVOTEK, Inc., all rights reserved EVT

29 Appendix C Preparation and Handling of PCR Samples With Wax For Thermal Cyclers without Heated Lids, or PCR Using Three Waterbaths 29 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. EDVOTEK, Inc. Copyright 2006, 2011, 2012 EDVOTEK, Inc., all rights reserved EVT

30 30 Appendix D 1.0% Agarose Gel Preparation If preparing the gel with concentrated (50x) buffer, use Table A.1. Table A.1 Individual 1.0% Agarose Gel Table A.2 If preparing the gel with diluted (1x) buffer, use Table A.2. Individual 1.0% Agarose Gel Size of Gel (cm) 7 x 7 Amt of Agarose (g) Concentrated Buffer (50X) (ml) Distilled Water (ml) 24.5 = Total Volume (ml) 25 Size of Gel (cm) 7 x 7 Amt of Agarose (g) Diluted Buffer (1x) (ml) 25 7 x x For DNA analysis, the recommended electrophoresis buffer is Tris-acetate-EDTA, ph 7.8. The formula for diluting EDVOTEK (50x) concentrated buffer is one volume of buffer concentrate to every 49 volumes of distilled or deionized water. Prepare buffer as required for your electrophoresis unit. Table B EDVOTEK Model # M6+ M12 M36 Electrophoresis (Chamber) Buffer Total Volume Dilution Required (ml) x Conc. Buffer (ml) Distilled Water (ml) Table C Volts Time and Voltage (1.0% - 7 x 7 cm gel) Recommended Time Minimum Maximum 30 min 45 min 90 min 45 min 60 min 120 min Time and Voltage recommendations for EDVOTEK equipment are outlined in Table C. The approximate time for electrophoresis will vary from approximately 2-3 hours depending upon various factors. Conduct electrophoresis for the length of time determined by your instructor. EDVOTEK, Inc. Copyright 2006, 2011, 2012 EDVOTEK, Inc., all rights reserved EVT

31 Appendix E 1.0% Agarose Gels - Quantity Preparations 31 To save time, electrophoresis buffer and agarose gel solution can be prepared in larger quantities for sharing by the class. Unused diluted buffer can be used at a later time and solidified agarose gel can be remelted. Table D Concentrated Buffer (50x) (ml) Bulk Preparation of Electrophoresis Buffer + Distilled Water (ml) Total Volume (ml) 60 2, (3 L) = BULK ELECTROPHORESIS BUFFER Quantity (bulk) preparation for 3 liters of 1x electrophoresis buffer is outlined in Table D. BATCH AGAROSE GELS (1.0%) For quantity (batch) preparation of 1.0% agarose gels, see Table E. Table E Amt of Agarose (g) Batch Preparation of 1.0% Agarose Concentrated Distilled + Buffer (50x) + Water = (ml) (ml) Total Volume (ml) 1. Use a 500 ml flask to prepare the diluted gel buffer 2. Pour the appropriate amount of Agarose into the prepared buffer. Swirl to disperse clumps. 3. With a marking pen, indicate the level of solution volume on the outside of the flask Note: The component Agarose is often labeled with the amount it contains. Please read the label carefully. If the amount of agarose is not specifi ed or if the bottle's plastic seal has been broken, weigh the agarose to ensure you are using the correct amount. 4. Heat the agarose solution as outlined previously for individual gel preparation. The heating time will require adjustment due to the larger total volume of gel buffer solution. 5. Cool the agarose solution to 60 C with swirling to promote even dissipation of heat. If evaporation has occurred, add distilled water to bring the solution up to the original volume as marked on the flask in step C 6. Dispense the required volume of cooled agarose solution for casting each gel. The volume required is dependent upon the size of the gel bed. 7. Allow the gel to completely solidify. It will become firm and cool to the touch after approximately 20 minutes. Then proceed with preparing the gel for electrophoresis. EDVOTEK, Inc. Copyright 2006, 2011, 2012 EDVOTEK, Inc., all rights reserved EVT

32 DNA InstaStain Patents Pending Patents Pending DNA InstaStain Patents Pending Patents Pending 32 Appendix F Staining and Visualization of DNA INSTASTAIN ETHIDIUM BROMIDE CARDS Do not stain gel(s) in the electrophoresis apparatus. 1. After electrophoresis, place the gel on a piece of plastic wrap on a flat surface. Moisten the gel with a few drops of electrophoresis buffer. 2. Wearing gloves, remove the clear plastic protective sheet, and place the unprinted side of the InstaStain EtBr card on the gel. 3. Firmly run your fingers over the entire surface of the InstaStain EtBr. Do this several times. Wear gloves and safety goggles 1 Moisten the gel. 2 DNA InstaStain Patents Pending DNA InstaStain Patents Pending 4. Place the gel casting tray and a small empty beaker on top to ensure that the InstaStain card maintains direct contact with the gel surface. Place the InstaStain card on the gel. Allow the InstaStain EtBr card to stain the gel for minutes. 5. After minutes, remove the InstaStain EtBr card. Transfer the gel to a ultraviolet (300 nm) transilluminator for viewing. Be sure to wear UV protective goggles. 3 Press firmly. 4 Visit our web site for an animated demonstration of InstaStain EtBr DNA InstaStain Caution: Ethidium Bromide is a listed mutagen. DNA InstaStain Place a small weight to ensure good contact. Disposal of InstaStain 5 Disposal of InstaStain cards and gels should follow institutional guidelines for chemical waste. Additional Notes About Staining If bands appear faint, gels may require longer staining with InstaStain EtBr. Repeat staining and increase the staining time an additional minutes. View on U.V. (300 nm) transilluminator DNA 200 bp markers should be visible after staining even if the amplifi ed DNA samples are faint or absent. If markers are not visible, troubleshoot for problems with the electrophoretic separation. EDVOTEK, Inc. Copyright 2006, 2011, 2012 EDVOTEK, Inc., all rights reserved EVT