BIOS 2011 Lab 5 PCR

BIOS 2011 Lab 5 PCR - Exercise 5 PCR PCR Detection of...

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Unformatted text preview: Exercise 5 PCR PCR Detection of Wolbachia Parasitic Bacterial Infection in Ants Learning Objectives: 1. To understand specimen collection and storage by collecting an ant specimen to test for infection. Ants of the genera Camponotus and Tetramorium will be compared. 2. To use PCR to detect Wolbachia infection in ant specimens. 3. To learn about PCR and gel electrophoresis. 4. To understand the purpose of a positive control in an experiment. Readings: Hillis, Sadava, Heller, and Price, Principles of Life, 2012. Pages 178-179, 246-249. Assignment: Data and analysis write-up. See Appendix II. Worth 20 points. INTRODUCTION Wolbachia is a genus of gram-negative bacteria that are reproductive parasites of arthropods. Wolbachia forms intracellular infections. It is estimated that 20-75% of all insects are infected with one or more species of this bacteria; Wolbachia species also infect nematodes, spiders and mites. Both male and female insects become infected, but the bacteria are inherited only through eggs via the cytoplasm. Wolbachia actively manipulate the host’s reproductive cycle to increase the chances of producing infected progeny. Wolbachia interfere with their reproduction in a variety of ways, including parthenogenesis by infected females, feminization of males, altering development of genitalia and cytoplasmic incompatibility of sperm and eggs. In general, these effects promote spreading the bacteria through the population. From an evolutionary perspective, these effects create reproductively isolated populations, which makes Wolbachia a force in speciation. We will be using ants you collect to test for infection rates. Each student will collect one ant. Do this as soon as possible on a clear, warm day. Keep the specimen in a freezer or refrigerator until you bring it to lab. You need to bring in your ant the day of the Mitosis & Meiosis lab (October 24/26). You will perform the DNA extraction and PCR reactions during this lab period. By running a DNA gel on November 14/16 you will find out which ants were infected. Once all sections have collected their data, we’ll pool the data so that you can compare infection rates between two genera of ants Camponotus and Tetramorium using a basic statistical analysis. Exercise 5 PCR PROTOCOL 1: CAMPONOTUS & TETRAMORIUM ANT COLLECTION Half the students in each section will be provided with a Yellow colored 1.5mL Eppendorf tube, and half with an Orange 1.5mL Eppendorf tube. Each tube contains ethanol, which will preserve the specimens. Put ONLY ONE ant in your Eppendorf tube. Try to avoid crushing the abdominal segment of the ant on the rear (posterior) end. Use a permanent marker to label the tube with lab day/section and collector's initials. If you are given an Orange tube, collect a large carpenter ant of the genus Camponotus. These are large, mostly black ants, c. 5-20mm long. Refer to pictures for their appearance and size. The Camponotus worker and soldier females are often found near or on trees or damp wood, and in old wooden buildings. Queens and males have wings but are only found when colonies divide by swarming, not often occurring when you are collecting. If you are given a Yellow tube, collect a small pavement ant of the genus Tetramorium. These are tiny, brown to dark brown ants that are only 3-5mm long. Refer to pictures for their appearance and size. Tetramorium are often around sidewalks and building foundations and near trees and shrubs. They can be attracted to a small container of sugar or food left outside. Again only queens and males have wings, but are not often available to be collected. Exercise 5 PCR Exercise 5 PCR PROTOCOL 2: ISOLATING AND PURIFYING INSECT DNA FOR PCR AMPLIFICATION As a first step to identifying a Wolbachia infection, you need to isolate DNA from the area of the insect’s body that is likely to harbor the bacteria. Ant gonads, which may have infected cells, are located in the most posterior body segment: the abdomen. The general procedure for isolating DNA, shown in the figure below, is to macerate (grind) the abdomen and lyse the cells and then separate DNA from other molecules by taking advantage of its negative charge and binding it to a chromatographic filter in a spin column. Other organic molecules are washed away by centrifuging liquids through the filter, and finally the purified DNA is eluted and spun out of the filter into an Eppendorf tube in the final spin. Carefully follow the steps in the flowchart below to purify DNA from the abdomen of the ant that you collected. Exercise 5 PCR PCR AMPLIFICATION SPECIFIC DNA SEQUENCES Now that we have a sample of DNA, we need to make many copies (or amplify) regions specific to insects and Wolbachia (to determine whether or not the insect is infected) in order to have enough DNA to visualize on a gel or sequence. We do this using the polymerase chain reaction, or PCR. Kary Mullis invented PCR about 25 years ago and was awarded the Nobel Prize in Chemistry for his invention in 1993. PCR is now a ubiquitous technique in the life sciences. This technique takes advantage of the fact that if you vary temperature, DNA strands will separate at one temperature and reanneal at another. Scientists combine their DNA samples with a mix of nucleotides and primers for a specific region, and Taq DNA polymerase. Taq polymerase comes from the bacteria Thermus aquaticus, which lives in hot springs and hydrothermal vents, and so has enzymes that function at high temperatures. The PCR process starts with heating the to 94-95˚C to denature and separate the template DNA strands of the original sample. The sample is cooled to 55-65˚C to allow short DNA primers to bind. Then the sample is heated to 72˚C to allow Taq to synthesize new DNA strands in the region defined by the primers. These steps are repeated a number of times to generate many copies derived from the desired template DNA region, which can then be observed on a gel. You will amplify DNA from the gene that codes for the Wolbachia surface protein (WSP ) to determine if your ant is infected. WSP is an outer membrane protein that is strongly expressed in Wolbachia. You will also amplify a section of mitochondrial DNA that codes for a “barcode” region of mitochondrial DNA found in insects but not in bacteria. Mitochondrial DNA is used as to differentiate among eukaryotic species because it shows relatively small amounts of variation within species and relatively large amounts of variation among species. Though we will not do it in this lab, this barcode DNA could be sequenced to identify the exact species of Camponotus or Tetramorium of your ant. However, we will simply be using the presence or absence of insect DNA as a positive control to make sure your DNA extraction and PCR reaction worked. Exercise 5 PCR PROTOCOL 3: PCR AMPLIFICATION Wear gloves. Use a clean pipette tip each time you transfer primers. You have isolated 200 µL of DNA in buffer. You will receive 4 other small Eppendorf tubes. Two clear 0.5 mL PCR Eppendorf tubes, each with a small white PCR bead. (1 set per student) One Purple 0.5 ml Eppendorf tube: WSP primer (1 tube per pair of students) One Green 0.5 ml Eppendorf tube: Insect barcode primer (1 tube per pair of students) The WSP primer identifies any Wolbachia infection. The insect barcode primer is used as a positive control to make sure you really extracted DNA from your ant. The small white thing in two of the Eppendorf tubes is the PCR bead. It contains the Taq DNA polymerase enzyme that catalyzes the DNA synthesis, the nucleotides, buffer, and magnesium chloride, necessary for the PCR reaction to occur. 1. Label the top of both PCR tubes with your with your initials and ant (C or T) type. Label one of these tubes for insect primer (I) and one for Wolbachia primer (W). 2. Add 22.5 µL of Insect primer mix to your ‘I’ PCR tube. Add 22.5 µL of WSP primer mix to your ‘W’ PCR tube. Pipette the liquids directly onto the bead, which will start to dissolve. 3. Add 2.5 µL DNA to both PCR tubes. 4. Close the caps and vortex the mix for 10 seconds. Centrifuge 30 seconds at maximum speed. When placed in the centrifuge rotor, these 0.5 mL tubes need to be put into a larger tube (such as a discarded collection tube from the DNA extraction) or they may become stuck during the spin. 5. Place your PCR tubes beside each other in the cold thermal cycler. Your TF will set the Thermal Cycler to 4˚C until everyone in the class has loaded their samples. After all samples are loaded your TF will start the thermal cycling program. Exercise 5 PCR The thermal cycler will be pre-programmed to carry out the following temperature cycles: 1 Genomic DNA denaturation 94˚C = 2 min 2 Thermal cycling (35 times) DNA denaturation 60 sec Primer binding 55˚C 60 sec DNA synthesis 3 94˚C 72˚C 60 sec Final extension 72˚C = 10 min Your samples will be frozen after amplification so that you can run a gel next lab. CHARACTERIZING PCR PRODUCTS USING GEL ELECTROPHORESIS During the Lac Operon lab you will also run your PCR products out on an agarose gel. Presence of WSP DNA indicates that your insect was infected with Wolbachia. The experimental control is the presence of insect DNA on your gel. If insect DNA is absent from your gel, it indicates there was a methodological error somewhere along the line, and that your Wolbachia result must be discarded. We will use a pre-cast agarose gel. For those of you who have run gels before, these gels do not require a buffer, and the DNA is stained with a fluorescent molecule . PROTOCOL 4: GEL ELECROPHORESIS OF PCR PRODUCTS 1. Plug in the E-Gel iBase. Do not remove the gel from the package more than 15 minutes before you want to run your samples. 2. Remove the gel from the package and insert the gel, with the comb in place, into the gel base right edge first. The comb should be at the top of the gel. Press firmly at the top and bottom to seat the gel in the base. You should hear a click when the gel is firmly seated. 3. Your TF will pre-run the gel 2 minutes. 4. Remove the comb by pulling straight up to expose the 12 wells. Exercise 5 PCR 5. Wearing gloves, load 20 ml of sample into each of two wells – the first should be your insect primer reaction, and the second should be your Wolbachia reaction. RECORD YOUR SAMPLE ID IN THE GEL DATA SHEET. 6. Your TF will start a program to run the gel for 30 minutes. 7. At the end of the run, unplug the power supply. Your TF will show you how to view the gels using a UV light source. BE SURE TO CLOSE THE LIGHT SHIELD BEFORE TURNING ON THE UV LIGHT. 8. Record whether or not your ant was infected with Wolbachia. How were you able to determine this? APPENDIX I. THE CHI-SQUARED TEST FOR HOMOGENEITY Reference URL: http://stattrek.com/AP-Statistics-4/Homogeneity.aspx?Tutorial=Stat In the case of the Ant / Wolbachia PCR lab, you have run a reaction with two sets of primers. One primer set is specific to the insect host organism, the ant, and should always appear when you run your gel. The second set of primers is specific to the bacteria and you will only see a band for this reaction if your ant was infected. The statistical test used for this purpose is the Chi-Squared Test of Homogeneity, with hypotheses: H0 (the null hypothesis): The number of infected ants from Genus A equals the number of infected ants from Genus B. (Ant populations A and B are homogeonous with respect to Wolbachia infection) H1 (the alternate hypothesis): The ant populations from Genus A and Genus B are not homogeneous with respect to infection by Wolbachia. See the Tutorial on Using the X2 test for Homogeneity on the course website for an example with all calculations shown. Before you begin the data analysis for this lab, go through the Practice Data presented below: Exercise 5 PCR Category 1 Category 2 Infected Uninfected Totals Population A 5 30 35 Population B 20 10 30 Totals 25 40 65 Table 1. Practice data for X2 Test for Homogeneity You need to calculate several variables from this data: Degrees of Freedom (DF) = (r-1) X (c-1) Where r = the number of populations being analyzed and c = the number of categories into which each population is divided. What is your DF value for this experiment? Expected Frequency count: E r, c = nr X nc/n Where nr = the number of observations from one of the populations, nc = the number of observations from both populations of one of that category, n= the total number of observations in your sample data set. So, for EA, 1 (the expected count for population A that are infected) = 35 X 25/65 = 13.46 You are using your data set to make a prediction about the entirety of both populations of ants. You need to calculate three other E values (population A, Uninfected; population B, Infected; Population B, Uninfected). The X2 value for this experiment will be the sum of four terms, which are described below. The chi-squared value equation is: X2 = Σ [ (Or,c - Er,c)2 / Er,c ] Where the example of EA, I has been defined above for you, and in this case the observed value (OA, I ) = 5. You will need to sum the values of (Or,c - Er,c)2 / Er,c for all four population categories to calculate X2 . Exercise 5 For this experiment, we will use the significance value of 0.05. PCR If the X2 value obtained is larger than the critical value of the X2 distribution for DF=1, and significance value = 0.05 (see table below), then you must reject the null hypothesis, and conditionally accept the alternate hypothesis. Do all the Practice Data calculations, refer to Table 2 for your critical value, and decide if you will accept or reject the null hypothesis. The actual class data on Wolbachia infection will be analyzed in exactly the same way as the Practice and Tutorial Data. Significant Probability for Critical Values df 0.995 0.975 0.9 0.5 0.1 0.05 0.025 0.005 1 0.000 0.000 0.016 0.455 2.706 3.841 5.024 7.879 2 0.010 0.051 0.211 1.386 4.605 5.991 7.378 10.597 3 0.072 0.216 0.584 2.366 6.251 7.815 9.348 12.838 4 0.207 0.484 1.064 3.357 7.779 9.488 11.143 14.860 Table 2. Distribution of Critical X2 Values for Degrees of freedom 1-4 Exercise 5 PCR APPENDIX II. GUIDELINES FOR THE LAB WRITEUP 1. Make a table showing the Numbers of Infected and Uninfected ants of each Genus, and the percentage infected in each Genus. This data will be made available to you electronically, emailed and on the web site. Write a table #, title and caption. 2. Describe the results in a paragraph that addresses the outcome of the experiment. Which hypothesis did your data support? 3. Include a discussion paragraph that addresses the following questions and any other ideas that you think are relevant to the experiment: How could the experiment be refined? What are other ways of grouping ants to look for differences in infection rates? 4. Include an appendix with your complete X2 calculations, tables, expected values, etc. Exercise 5 LAB WRITEUP CHECKLIST AND GRADESHEET: PCR 20 points total PCR Table #1 – (5 points) This table should include:  ­ Numbers of infected and uninfected ants of each genus and the percentage infected in each genus.  ­ Table #  ­ Title  ­ A complete caption that:  ­ Describes the result of the experiment.  ­ Clearly states the hypotheses to be tested. Discussion Paragraph – (10 points) This paragraph should include:  ­ What the outcome of the experiment was.  ­ Which hypotheses was supported or refuted.  ­ A discussion of how the experiment could be refined.  ­ A discussion of how the ants could be grouped to look for differences in infection rate.  ­ Any other ideas that you feel are relevant to the experiment. Appendix – (5 points) The appendix should include:  ­  ­ Chi-squared values obtained  ­ Expected Frequency Counts  ­ Chi-squared calculation Expected Critical Chi-Squared Value ...
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