RFLP analysis and agarose gel electrophoresis

Transcription

1 RFLP analysis and agarose gel electrophoresis Purpose In this lab you will be examining the safety concerns in one type of very commonly used molecular biology procedure: agarose gel electrophoresis. You will run a mock DNA analysis called a restriction fragment length polymorphism or RFLP analysis. This type of experiment requires the pouring, running and staining of agarose gels. You will Use Safety Data Sheets to investigate the chemical hazards associated with agarose gel electrophoresis Identify the various hazards associated with pouring, running, staining, and viewing agarose gels Learn how to prepare agarose gels Learn how to safely work with ethidium bromide solutions Understand the basics of RFLP analysis Background Restriction Length Polymorphism (RFLP) analysis RFLP analysis is a way to differentiate between the DNA of various species or between individuals of the same species. Essentially, an RFLP analysis involves cutting up the genome (which is all the DNA present in a single cell of a particular organism) into pieces of various sizes that can be separated on an agarose gel. The pattern of sizes that is visualized on the gel will be a pattern that is unique to that individual or species. (See Figure 1.) The uses of RFLP analysis are numerous. They include identification tests in criminal cases and studies on how similar organisms are to one another at the DNA level. In this lab, we will use a mock RFLP analysis to differentiate between two types of viruses. 1

2 Figure 1: A B C DNA Cut DNA into pieces A B C Separate by size on agarose gel Well of DNA gel B A C 2

3 The major steps in RFLP analysis DNA from an organism is isolated and cut into pieces with an enzyme called a restriction enzyme. The pieces will vary in number and size with species or individual. Here, a restriction enzyme has cut the genome into fragments A, B, and C, which vary in size. These fragments can be separated by size on an agarose gel, in which the largest fragment (B), runs slowest, and the smallest (C) runs the fastest. The pattern of fragments viewed on the gel will help with identification of species or individual. Agarose gel electrophoresis basics Agarose gel electrophoresis is one of the most commonly used techniques in molecular biology. This procedure is a method to separate DNA molecules based upon their size. Agarose is a polysaccharide that may be dissolved in a buffer containing Tris, Boric Acid and EDTA. This buffer is called TBE. Upon heating of the agarose/tbe mixture, the agarose dissolves. This mixture is then poured into a casting tray. Upon cooling, the agarose solidifies into a gel matrix that will act as a sieve for the DNA molecules. DNA is added to the gel matrix by using a micropipet to load a DNA/ loading dye mixture into depressions in the gel, called wells. The agarose gel is run in an electrophoresis tank that is filled with TBE buffer. An electrical current is applied across the gel. DNA is highly negatively charged, and will, therefore, run towards the positive electrode. As the DNA molecules pass through the agarose matrix, the molecules separate based, primarily, upon their size. The smallest DNA molecules traverse the gel the most quickly, while the largest molecules have a more difficult time passing through the gel matrix. In order to visualize the DNA molecules within the gel matrix, the gel is stained with a dye called ethidium bromide. This dye fluoresces under ultraviolet light. When the gel is placed in an ethidium bromide solution, the dye binds to the DNA in the gel. When the stained gel is placed on a transilluminator, the DNA molecules will fluoresce. The mock experimental scenario In March 2003, a 35 year-old man was diagnosed with Fifth Disease (which is caused by a parvovirus) at a hospital in Albuquerque, NM. He was sent home to recover, but later that same evening, his symptoms rapidly worsened and he returned to the hospital emergency room. He was admitted to the hospital, where he received ten days of care until he was well enough to be sent home to finish his recovery. Over the course of the next two months, 3000 similar cases were diagnosed in the United States, resulting in 152 deaths. Although the initial symptoms of this illness very closely mimicked that of the virus that causes Fifth Disease, a common childhood illness that has few complications, the rapid progression and high death rate indicated that this was not actually a commonly found parvovirus. The Centers for Disease Control and Prevention became involved very early in the development of this potential epidemic. Scientists were able to isolate the virus from patients. They compared the genome of the parvovirus (causing Fifth Disease) to that of this potentially new and lethal virus using RFLP analysis. They found that they could rapidly differentiate between the Fifth Disease virus and this new strain of virus using this technology. 3

4 Laboratory activity Overview: You are working in a diagnostic laboratory. You are ready to run a gel on a previously prepared RFLP analysis. You are given the following samples: Sample 1: Control RFLP analysis from Fifth Disease virus Sample 2: Control RFLP analysis from new virus Sample 3: RFLP analysis from patient 1 w/ Fifth Disease symptoms Sample 4: RFLP analysis from patient 2 w/ Fifth Disease symptoms Sample 5: RFLP analysis from patient 3 w/ Fifth Disease symptoms Your goal is to use agarose gel electrophoresis to compare the RFLP results from these three patients to determine which, if any, of the three are likely to develop the new, potentially fatal illness rather than show the normal progression of Fifth Disease. This lab will consist of the following parts: Investigation of Safety Data Sheets Preparation of an agarose gel Loading of the gel Electrophoresis of the gel Staining of the gel with ethidium bromide Photography of the stained gel Analysis of RFLP data and diagnosis Experimental protocol PART ONE: Investigation of Safety Data Sheets Examine the Safety Data Sheets for the following chemicals and record any precautions to be taken in your lab notebook: Tris EDTA Boric Acid Ethidium bromide PART TWO: Preparation of a 1% agarose gel 1. Write out the procedure to make a 1% (w/v) agarose gel in TBE. You will need 30 ml of agarose for your gel. 2. Weigh out the amount of agarose you calculated in step 1 and place it in a 100 ml flask. 3. Add 30 ml of TBE to the flask. 4. Microwave the flask containing the TBE/agarose mixture for about 45 seconds. WATCH THE FLASK WHILE MICROWAVING SO THAT IT DOES NOT BOIL OVER. 5. Remove the flask from the microwave USING HEAT-RESISTANT GLOVES and gently swirl the flask. If you can still see agarose particles, return the flask to the microwave for about 15 more seconds. 6. Allow the agarose solution to cool to about 50 degrees Celsius. 7. Pour cooled agarose into casting tray with comb as demonstrated by your instructor. 8. Allow gel to solidify. 4

5 PART THREE: Loading and electrophoresis of the agarose gel 1. Loading dye has been added to your DNA samples. This dye will allow visualization of how far the DNA has run on the gel and will help the DNA sink into the wells. 2. Remove the comb from the solidified gel. 3. Place the gel, in its casting tray, into the electrophoresis tank. Make sure you put down the gates of the casting tray. 4. Add TBE to the tank so that the surface of the gel is just covered. 5. Load your DNA samples into the wells of the gel as demonstrated by your instructor. RECORD WHICH SAMPLE IS LOADED INTO EACH LANE IN YOUR LABORATORY NOTEBOOK. 6. Connect the leads of the electrophoresis apparatus to the power supply. 7. Run the gel at 120 V for about 40 minutes. PART FOUR: Staining and photographing the agarose gel 1. Turn off the power supply. 2. Disconnect the leads and remove the casting tray and gel from the electrophoresis tank. 3. Put on gloves. 4. Slide the gel from the casting tray into a Tupperware container set aside for ethidium bromide use. DO NOT PLACE THE CASTING TRAY OR ANYTHING ELSE DOWN ON THE BENCH RESERVED FOR ETHIDIUM BROMIDE USE. ALSO, DO NOT TOUCH THE CASTING TRAY TO THE ETHIDIUM BROMIDE STAINING TRAY. 5. Add ethidium bromide staining solution to just cover the gel in the tray. 6. Stain for 10 minutes with gentle agitation. 7. Using a funnel, carefully pour the ethidium bromide solution back into the stock bottle. 8. Using a spatula, remove the gel from the staining tray and place it on a UV light box. 9. Photograph the picture as demonstrated by your instructor. 10. Discard your gel in the appropriate waste beaker. Results: 1. Label the lanes of your picture. 2. Compare the results of patients 1 through 3 with the two control samples. 3. Record in your notebook which of the patients is likely to be infected with which virus. Discussion questions 1. From your study of the Safety Data Sheets, what did you discover about the risk associated with the use of ethidium bromide? 2. What risks are associated with preparation of agarose gel? What precautions help to minimize these risks? 3. What risks are associated with running the agarose gel? What precautions help to minimize these risks? 4. What risks are associated with viewing of the ethidium bromide-stained gel? What precautions help to minimize these risks? 5. List all special precautions used when working with ethidium bromide. 5