S-54. What is qpcr and How Does It Work? NEW. Edvo-Kit #S-54. Experiment Objective: See page 3 for storage instructions.

Transcription

1 NEW Edvo-Kit #S-54 What is qpcr and How Does It Work? S-54 Experiment Objective: This experiment explores the principles of qpcr by analyzing colorful dye samples using agarose gel electrophoresis. Students will observe the relationship between cycle number and amount of DNA present within a sample. See page 3 for storage instructions. S

2 What is qpcr and How Does It Work? EDVO-Kit S-54 Table of Contents Page Experiment Components 3 Experiment Requirements 3 ackground Information 4 Experiment Procedures Experiment Overview 7 Agarose Gel Electrophoresis 9 Study Questions 11 Instructor's Guidelines 12 Pre-Lab Preparations 13 Experiment Results and Analysis 14 Study Questions and Answers 15 Appendices 16 Material Safety Data Sheets can be found on our website: EDVOTEK and The iotechnology Education Company are registered trademarks of EDVOTEK, Inc. Ready-to-Load, QuickStrips and UltraSpec-Agarose are trademarks of EDVOTEK, Inc. 2

3 EDVO-Kit S-54 What is qpcr and How Does It Work? Experiment Components READY-TO-LOAD SAMPLES OR ELECTROPHORESIS Store all components at room temperature. Components (in QuickStrip format) Check ( ) A Standard dyes with assigned base pair equivalents Sample after 10 cycles C Sample after 20 cycles D Sample after 30 cycles E Sample after 40 cycles REAGENTS & SUPPLIES UltraSpec-Agarose Electrophoresis uffer (50x) Practice Gel Loading Solution 1 ml pipet Microtipped Transfer Pipets Experiment #S-54 is designed for 10 gels. The QuickStrip samples are stable at room temperature for up to one month after receipt. However, if the experiment will not be conducted within this time frame, it is recommended that the QuickStrip samples be stored in the refrigerator. Requirements Horizontal gel electrophoresis apparatus D.C. power supply Automatic micropipets with tips alance Microwave, hot plate or burner Pipet pump 250 ml flasks or beakers Hot gloves Safety goggles and disposable laboratory gloves DNA visualization system (white light) Distilled or deionized water All experiment 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. 3

4 What is qpcr and How Does It Work? EDVO-Kit S-54 ackground Information THE POLYMERASE CHAIN REACTION (PCR) In 1984, Dr. Kary Mullis revolutionized the field of molecular biology when he devised a simple and elegant method to copy specific pieces of DNA. Recognizing that an initial step in DNA replication in a cell s nucleus is the binding of RNA primers, Mullis discovered that he could replicate DNA in vitro using short, synthetic DNA primers and DNA polymerase I. urthermore, because researchers can specify a primer s sequence to target a specific gene, this method allowed for the rapid amplification of a selected DNA sequence in vitro. or the development of this technique, known today as the Polymerase Chain Reaction (or PCR), Mullis was awarded the Nobel Prize in Target Sequence Chemistry in In order to amplify DNA, purified double-stranded DNA is mixed with the short DNA primers, a thermostable DNA polymerase (Taq) and nucleotides. The mixture is heated to 94 C to denature (i.e., unzip into single strands by breaking hydrogen bonds) the DNA duplex. Next, the sample is cooled to 45 C-60 C, allowing the primers to base pair with their target DNA sequences (a step known as annealing ). Lastly, the temperature is raised again, to 72 C, the optimal temperature at which Taq polymerase will extend the primer to synthesize a new strand of DNA. Each cycle (denaturation, annealing, extension) doubles the amount of target DNA (igure 1). Today, a specialized machine, called a thermal cycler or PCR machine, is used to rapidly heat and cool the samples. As a result, a PCR cycle can be completed in less than 5 minutes; cycles produce sufficient DNA for analysis. Cycle 1 Cycle 2 Cycle 3 = = = Separation of two DNA strands Primer 1 Primer 2 igure 1: The Polymerase Chain Reaction Denature 94 C Anneal 2 primers 45 C Extension 72 C 4

5 EDVO-Kit S-54 What is qpcr and How Does It Work? PRINCIPLES O QUANTITATIVE PCR The products of conventional PCR are most often analyzed by agarose gel electrophoresis. If a target DNA sequence is present in the starting material and is amplified by the PCR reaction, a band of DNA will be visible when the gel is stained (igure 2). Therefore, conventional PCR coupled with electrophoresis produces a yes/no qualitative result. In contrast, quantitative PCR (qpcr, also known as real-time PCR) can determine the exact amount of target DNA in the starting material by measuring the accumulation of DNA as the reaction progresses. Electrophoresis is not required because amplification and quantitation of the DNA occur simultaneously. Similarly to conventional PCR, each cycle of real-time PCR doubles the amount of the DNA in the sample (igure 1). Mathematically, this doubling can be expressed as an exponential relationship if we begin with a starting copy number of m, then after n cycles, we will have m x 2n copies of our DNA target. or example, if we start with one copy of our target, we will have two copies after the first PCR cycle, four after the second PCR cycle, eight after the third PCR cycle, and so on. After many cycles (regardless of the amount of DNA present in the starting material) the amount of DNA produced reaches a maximum where a product curve flattens out, known as the plateau (igure 3). This leveling off of the curve is due to the depletion of reaction components like primers and nucleotides and the loss of Taq polymerase activity. igure 2: Results of a conventional PCR experiment as analyzed by agarose gel electrophoresis. In contrast to conventional PCR, real-time PCR samples contain special fluorescent dyes that produce light when bound to double-stranded DNA (igure 4). This allows the user to measure the amount of DNA in a sample as it is being synthesized. The amplification is performed in a thermal cycler that can excite the fluorescent molecules and detect the signal that they produce. A measured increase in fluorescence directly relates to an increase in the amount of amplified DNA in the sample. In early cycles of PCR, fluorescence is low because there is not a lot of DNA present in the sample. As the number of cycles increases, the PCR product accumulates, and so fluorescence increases. The cycle during which the fluorescence reaches a set threshold is known as the quantification cycle, or Cq (igure 3). As the concentration of DNA template increases, the number of cycles it takes to reach the Cq decreases. or example, if the DNA template is present in the sample in low levels, it takes many cycles before the fluorescence can be detected (high Cq). Conversely, if the target DNA is abundant in the starting material, the fluorescence will increase to measurable levels relatively quickly (low Cq). The exact number of target DNA molecules in a sample can be determined by comparing its Cq value to those from samples of known concentration using a standard curve. To create a standard curve, a DNA template is diluted over several orders of magnitude (for example, from microgram to picogram quantities), and the Cq is determined for each sample (igure 5). Plotting Cq on the y-axis and the log 10 of the known DNA concentration on the x-axis results in a straight line. The equation of this line is used to determine the starting concentration of our unknown sample by substituting the measured Cq value into the equation. luorescence Threshold line Exponential phase Cq value Non Exponential plateau phase igure 3: Graph showing the exponential phase and plateau phase of PCR. 5

6 What is qpcr and How Does It Work? EDVO-Kit S-54 APPLICATIONS O QPCR TECHNOLOGY Real-time PCR is a commonly used technique in the both the research and the diagnostic laboratory because it is fast, sensitive, and requires less material and technical skill than traditional techniques like Northern or Southern blotting. or example, microbiologists commonly use qpcr to both identify and quantify microorganisms in food and water samples. Physicians may use qpcr to establish the exact level, or titer, of a particular bacteria or virus present in a specific patient sample. ecause qpcr can differentiate between specific strains of a particular pathogen (like influenza A and ), it is a powerful diagnostic and informational tool for health professionals. A. Denaturation Step Taq. Annealing Step Taq qpcr can also be used to determine the extent that a specific gene is turned on, i.e., how much RNA is being transcribed from that particular gene. irst, the RNA must be converted into DNA before it can be quantified. This process, known as reverse transcription, creates a complementary DNA (cdna) sequence from an RNA template. Once the cdna is produced, qpcr can be used to quantify the amount of cdna and, by extension, the amount of original RNA present in the sample. This is very useful when biotechnology companies need to determine the effects of experimental medications on specific biological pathways. or these reasons, qpcr has become an essential technique for today's scientists. C. Extension Step igure 4 This experiment explores the principles of qpcr using colorful dye samples. Using agarose gel electrophoresis, they will observe the relationship between cycle number and amount of DNA present within a sample. Students will perform data analysis to support these observations. Quantification Cycle Correlation Coefficient: Slope: Intercept: Y= X= Standards Unknowns Log Starting Quantity, copy number igure 5 6

7 EDVO-Kit S-54 What is qpcr and How Does It Work? Experiment Overview EXPERIMENT OJECTIVE: This experiment explores the principles of qpcr by analyzing colorful dye samples using agarose gel electrophoresis. Students will observe the relationship between cycle number and amount of DNA present within a sample. LAORATORY SAETY 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. 5. Always wash hands thoroughly with soap and water after handling reagents or biological materials in the laboratory. LAORATORY NOTEOOKS: Scientists document everything that happens during an experiment, including experimental conditions, thoughts and observations while conducting the experiment, and, of course, any data collected. Today, you ll be documenting your experiment in a laboratory notebook or on a separate worksheet. efore starting the Experiment: Carefully read the introduction and the protocol. Use this information to form a hypothesis for this experiment. Predict the results of your experiment. During the Experiment: Record your observations. After the Experiment: Interpret the results does your data support or contradict your hypothesis? If you repeated this experiment, what would you change? Revise your hypothesis to reflect this change. 7

8 What is qpcr and How Does It Work? EDVO-Kit S-54 Experiment Overview 2 Remove end blocks & comb, then submerge gel under buffer in electrophoresis chamber 1 Prepare agarose gel in casting tray 3 Load each dye sample in consecutive wells A C D E 4 Attach safety cover,connect leads to power source and conduct electrophoresis After electrophoresis, transfer gel for visualization. 5 Analysis on white light source ( - ) Gel pattern will vary depending upon the experiment. 8

9 EDVO-Kit S-54 What is qpcr and How Does It Work? Agarose Gel Electrophoresis x Concentrated buffer lask Distilled water Agarose 1:00 Caution! lask will be HOT! IMPORTANT: If you are unfamiliar with agarose gel prep and electrophoresis, detailed instructions and helpful resources are available at C Wear gloves and safety goggles C Pour WAIT 20 min DILUTE concentrated (50X) buffer with distilled water to create 1X buffer (see Table A). 2. MIX agarose powder with 1X buffer in a 250 ml flask (see Table A). 3. DISSOLVE agarose powder by boiling the solution. MICROWAVE the solution on high for 1 minute. Carefully REMOVE the flask from the microwave and MIX by swirling the flask. Continue to HEAT the solution in 15-second bursts until the agarose is completely dissolved (the solution should be clear like water). 4. COOL agarose to 60 C with careful swirling to promote even dissipation of heat. 5. While agarose is cooling, SEAL the ends of the gel-casting tray with the rubber end caps. PLACE the well template (comb) in the appropriate notch. 6. POUR the cooled agarose solution into the prepared gel-casting tray. The gel should thoroughly solidify within 20 minutes. The gel will stiffen and become less transparent as it solidifies. 7. REMOVE end caps and comb. Take particular care when removing the comb to prevent damage to the wells. Table A Size of Gel Casting tray 7 x 7 cm 7 x 10 cm Individual 0.8% UltraSpec-Agarose Gel Concentrated uffer (50x) 0.6 ml 1.0 ml + Distilled Water ml 49.0 ml Amt of Agarose 0.23 g 0.39 g = TOTAL Volume 30 ml 50 ml 7 x 14 cm 1.2 ml 58.8 ml 0.46 g 60 ml 9

10 What is qpcr and How Does It Work? EDVO-Kit S-54 Agarose Gel Electrophoresis Pour 1X Diluted uffer Reminder: efore loading the samples, make sure the gel is properly oriented in the apparatus chamber ( - ) ( + ) 8. PLACE gel (on the tray) into electrophoresis chamber. COVER the gel with 1X electrophoresis buffer (See Table for recommended volumes). The gel should be completely submerged. 9. LOAD the entire sample (35-38 μl) into the well in consecutive order. The identity of each sample is provided in Table PLACE safety cover. CHECK that the gel is properly oriented. Remember, the DNA samples will migrate toward the positive (red) electrode. 11. CONNECT leads to the power source and PERORM electrophoresis (See Table C for time and voltage guidelines). 12. After electrophoresis is complete, REMOVE the gel and casting tray from the electrophoresis chamber and VISUALIZE the results. No staining is necessary. Lane Tube A Tube Tube C Tube D Tube E Table 1: Gel Loading Standard dyes with assigned bp equivalents Sample after 10 cycles Sample after 20 cycles Sample after 30 cycles Sample after 40 cycles Table EDVOTEK Model # M6+ M12 M36 1x Electrophoresis uffer (Chamber uffer) Total Volume Required 300 ml 400 ml 1000 ml 50x Conc. uffer 6 ml 8 ml 20 ml Dilution + Distilled Water 294 ml 392 ml 980 ml Table C Volts Time and Voltage Guidelines (0.8% Agarose Gel) Electrophoresis Model M6+ M12 & M36 Min. / Max. Min. / Max. 15/20 min. 20/30 min. 35 / 45 min. 25 / 35 min. 35 / 45 min. 60 / 90 min. 10

11 EDVO-Kit S-54 What is qpcr and How Does It Work? Study Questions 1. List and describe the three basic steps of conventional PCR. 2. What are some differences between conventional PCR and real-time PCR? 3. What is the Cq? How is the Cq related to the initial DNA concentration? 4. Imagine that you are a physician and you are trying to treat bacterial infection in three different patients. You decide to use qpcr to determine whether or not the treatment is effective. A high bacterial load (over 10 pg of bacterial DNA) suggests that the infection is not being cleared and the treatment must change, whereas a low bacterial load (less than 10 pg of bacterial DNA) suggests treatment is working. elow is the data from qpcr test. Using the standard curve, determine the bacterial load of the three patients. Which patients are being treated effectively? Which patients need a different treatment? Patient 1 Cq = 25 Patient 2 Cq = 18 Patient 3 Cq = Quantification Cycle DNA (pg) 11

12 INSTRUCTOR'S GUIDE What is qpcr and How Does It Work? EDVO-Kit S-54 Instructor's Guide OVERVIEW O INSTRUCTOR S PRELA PREPARATION: This section outlines the recommended prelab preparations and approximate time requirement to complete each prelab activity. What to do: When: Time Required: Prepare QuickStrips Prepare diluted TAE buffer Up to one day before performing the experiment. 40 min. Prepare molten agarose and pour gel 12 EDVOTEK, Inc., all rights reserved. S

13 EDVO-Kit S-54 What is qpcr and How Does It Work? INSTRUCTOR'S GUIDE Pre-Lab Preparations: Module I SEPARATION O PCR PRODUCTS Y AGAROSE GEL ELECTROPHORESIS This experiment requires a 0.8% agarose gel per student group. You can choose whether to prepare the gels in advance or have the students prepare their own. Allow approximately minutes for this procedure. Individual Gel Preparation: Each student group can be responsible for casting their own individual gel prior to conducting the experiment. See Module I in the Student s Experimental Procedure. Students will need 50x concentrated buffer, distilled water and agarose powder. atch Gel Preparation: To save time, a larger quantity of agarose solution can be prepared for sharing by the class. See Appendix. NOTE: Accurate pipetting is critical for maximizing successful experiment results. EDVOTEK Series 100 experiments are designed for students who have had previous experience with micropipetting techniques and agarose gel electrophoresis. If students are unfamiliar with using micropipets, we recommended performing Cat. #S-44, Micropipetting asics or Cat. #S-43, DNA DuraGel prior to conducting this advanced level experiment. Preparing Gels in Advance: Gels may be prepared ahead and stored for later use. Solidified gels can be store under buffer in the refrigerator for up to 2 weeks. Do not freeze gels at -20º C as 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 molten agarose before being placed into the tray. This will prevent the gels from sliding around in the trays and the chambers. OR MODULE I Each Student Group should receive: 50x concentrated buffer Distilled Water UltraSpec-Agarose Ready-to-Load Samples SAMPLES ORMAT: PREPARING THE QUICKSTRIPS QuickStrip tubes consist of a microtiter block covered with a protective overlay. Each well contains pre-aliquoted dyes. Using sharp scissors, carefully divide the block of tubes into individual strips by cutting between the rows (see diagram at right). Take care not to damage the protective overlay while separating the samples. EDVOTEK DO NOT END A C D E G CUT HERE A C D E G CUT HERE A C D E G CUT HERE A C D E G CUT HERE A C D E G CUT HERE A C D E G Each lab group will receive one set of tubes. efore loading the gel, remind students to tap the tubes to collect the sample at the bottom of the tube. H H H H H Carefully cut between each set of tubes H 13

14 INSTRUCTOR'S GUIDE What is qpcr and How Does It Work? EDVO-Kit S-54 Experiment Results and Analysis ,500 1, R P Y In this qpcr simulation, the amount of dye increases as it proceeds through the qpcr reaction cycles, as shown in Lanes 2 through 5 in the above figures. S-54 Gel Idealized schematic: The relative positions of dye molecules are shown but are not depicted to scale. Lane 1 A Standard dyes with assigned base pair equivalents = lue R = Red P = Purple Y = Yellow 2 Sample after 10 cycles 3 C Sample after 20 cycles 4 D Sample after 30 cycles 5 E Sample after 40 cycles 14

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

16 APPENDICES What is qpcr and How Does It Work? EDVO-Kit S-54 Appendices A EDVOTEK Troubleshooting Guide ulk Preparation of Agarose Gels Material Safety Data Sheets: Now available for your convenient download on 16 EDVOTEK, Inc., all rights reserved. S

17 EDVO-Kit S-54 What is qpcr and How Does It Work? APPENDICES Appendix A EDVOTEK Troubleshooting Guides PROLEM: CAUSE: ANSWER: ands not visible on the gel The electrophoresis buffer was not prepared properly. The dyes ran off of the gel because the polarity of the leads was reversed. Malfunctioning electrophoresis unit or power source. Ensure that the electrophoresis buffer was correctly diluted. Ensure that leads are attached in the correct orientation. Contact the manufacturer of the electrophoresis unit or power source. Very light colored band seen after electrophoresis Pipetting error. Make sure students pipet 35 µl of dye sample per well. Poor separation of bands Gel was not prepared properly. Make sure to prepare a 0.8% gel. Dye bands disappear when the gels are kept at 4 C. The dye molecules are small and will diffuse out of the gel. The results must be analyzed upon the completion of electrophoresis EDVOTEK, Inc., all rights reserved. S

18 APPENDICES What is qpcr and How Does It Work? EDVO-Kit S-54 Appendix ulk Preparation of Agarose Gels To save time, the 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 solution can be remelted. ulk Electrophoresis uffer Quantity (bulk) preparation for 3 liters of 1x electrophoresis buffer is outlined in Table D. Table D ulk Preparation of Electrophoresis uffer 50x Conc. uffer + Distilled Water Total Volume Required 60 ml 2,940 ml 3000 ml (3 L) atch Agarose Gels (0.8%) or quantity (batch) preparation of 0.8% agarose gels, see Table E. 1. Use a 500 ml flask to prepare the diluted gel buffer 2. Pour 3.0 grams of UltraSpec-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 UltraSpec-Agarose kit component is usually 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 and DNA staining method which will be used. Refer to Appendix A or for guidelines. 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. Table E Amt of Agarose (g) atch Prep of 0.8% UltraSpec-Agarose + Concentrated uffer (50X) (ml) + Distilled Water (ml) Total Volume (ml) EDVOTEK, Inc., all rights reserved. S