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TBIOL 130 Clark Atlanta University PCR Amplification Lab Report
Problem Set V Central Dogma TBIOL 130 (Winter ‘23) Name: Score: Answer the following questions to the best of your ability; this exercise is worth 30 points and must be uploaded to Canvas by midnight on Friday of Week 6 (Feb. 10th). You may work together with a group to complete this exercise, but everyone must submit their completed problem sets separately. Please note that “work together with a group” does not mean that you can submit identical answers. In other words, you can consult with your peer(s) on the answer (or the means to come up with the answer) but you need to answer the questions in your own words. You may provide your answers in a separate file or within this worksheet (save as a .pdf file) but for the latter, remember to TYPE YOUR ANSWERS IN RED (or another color of your choice)! Part I: Gene Regulation of the trp Operon The following questions are about the prokaryotic trp operon (outlined below), specifically addressing how the operon is regulated in response to nutritionally poor environments. Briefly, the trp operon includes a group of 5 genes (described below as genes A-E) that encode for the biosynthetic enzymes required to synthesize the amino acid, tryptophan. The structure of the trp operon is shown, including the promoter sequence which contains a regulatory sequence known as an operator. Use your knowledge of gene expression and consider the consequence for a bacterium in the following situations. 1. (4 points) The trp operon is expressed (i.e. turned ”on”) when cytoplasmic tryptophan levels are low, which allows the bacterium to synthesize tryptophan. However, the trp operon is repressed (i.e. turned “off”) when tryptophan levels are high. The cause of the repression is a bacterial protein known as the trp repressor. a. When the trp repressor is bound to tryptophan, the protein can readily recognize and bind to the operator sequence. Why would this action prevent the expression of the trp operon? b. How would a drop in the cytoplasmic levels of tryptophan allow for the expression of the trp operon? Hint: you will need to consider your answer to Q1A. Problem Set V Central Dogma TBIOL 130 (Winter ‘23) 2. (3 points) Given what you know about gene expression, consider how much “effort” the cell puts into expressing a gene product. How would the energy requirements necessary to synthesize a protein help explain why organisms would go through the effort of regulating gene expression? In other words, why wouldn’t a bacterium express the trp operon all the time (as opposed to only expressing it when tryptophan levels are low)? 3. (3 points) What would happen if there was a mutation that caused the trp repressor protein to bind more tightly to tryptophan (even if tryptophan levels were low)? 4. (3 points) What would happen if there was a mutation in the trp repressor binding sequence (the operator sequence) that made the repressor unable to bind (even if tryptophan levels were high)? 5. (3 points) What would happen to the cell (when tryptophan levels are low) and there was a nonsense mutation early in the coding sequence of gene D? 6. (4 points) What would happen if a mutation were introduced in the DNA binding domain of the Trp repressor that changed an asparagine residue to a glycine residue? Would this be more problematic for the cell when tryptophan levels were high or low? 7. (2 points) Do eukaryotes have operons? If not, explain how eukaryotes would regulate the expression of multiple related genes so that they are expressed at the same time. Problem Set V Central Dogma TBIOL 130 (Winter ‘23) Part II: Gene Regulation of the ara Operon In the absence of glucose, Escherichia coli can still generate the energy necessary for proliferation by using the pentose sugar arabinose. The figure below outlines the arabinose (ara) operon, which encodes for all the proteins necessary to achieve this switch in sugar metabolism. The araA, araB and araD genes encode the enzymes for the metabolism of arabinose while the araC gene encodes a transcription regulator that binds adjacent to the promoter of the arabinose operon. To understand the regulatory properties of the AraC protein, you engineer a mutant E. coli in which the araC gene has been deleted so that you can look at how the presence or absence of the AraC protein affects the expression of the AraA enzyme. 1. (4 points) If the AraC protein works as a repressor, would you expect araA RNA levels to be high or low in the presence of arabinose in the araC– mutant cells? What about in the absence of arabinose? Explain your answer. Your findings from the experiment are summarized in the following table. 2. (4 points) Do the results indicate that the AraC protein regulates arabinose metabolism by acting as a repressor or an activator? Explain your answer. Problem Set V Central Dogma TBIOL 130 (Winter ‘23) BONUS (1 point): Below is a picture of a transcription factor whose asparagine side chain (shown on the right) is contacting an adenine ring in a DNA molecule. a. Which of the amino acids shown below would be able to make the same H-bonding contacts as the Asparagine? Circle your answer. b. Would the substitution of the amino acid from part A change the strength of the protein-DNA complex? Why or why not? CATCHING CHEATERS Part 1: Salmon DNA Extraction/Purification *Remember to bring your salmon samples & completed sample information sheet/table to lab!!!* Learning Objectives ● ● ● ● Extract and purify DNA from salmon tissues by following a detailed protocol. Explain the basic biochemical principles behind DNA extraction/purification. Practice proper laboratory technique in pipetting and centrifugation. Practice accurate record keeping and sample labeling. Lab Preparation & Study Questions 1. Obtain 1-2 small salmon samples (in provided tubes/dishes) and complete sample information sheet. 2. Watch the “Isolating DNA from Salmon Tissue” video posted on Canvas. 3. Read the entire lab handout and be prepared to answer the following questions: a. Describe the difference between a vortexer and a centrifuge. What do each of these pieces of lab equipment do? b. How large should the salmon tissue be for this procedure? c. What is the purpose of the Proteinase K treatment? d. What is a pellet? What is a supernatant? Be able to define both. e. What is the DNeasy mini spin column? Be able to describe its appearance. f. What is the color of the final DNA solution? Introduction to DNA By now, you should all be familiar with proteins and enzymes, the biological macromolecules that control the almost infinite number of interactions and life processes in cells and living organisms. However, the molecular information that encodes these important proteins and enzymes is contained within the genome, the sequences of deoxyribonucleic acid or DNA that make up the blueprint of life found within each living organism. With amazing precision, this blueprint gets passed on from generation to generation of each species. Catching Cheaters I – Salmon DNA Extraction The DNA molecule is a long polymer consisting of four different nucleotides. Each nucleotide is composed of a deoxyribose sugar, a phosphate group, and a nitrogenous base (Adenine, Guanine, Tyrosine, or Cytosine). Nucleotide chains of DNA are formed when the phosphate group of one nucleotide is joined to the deoxyribose sugar of the next nucleotide, creating the sugar-phosphate “backbone” of the DNA molecule. The various combinations of the four nucleotide bases protruding from the backbone make up the unique DNA code or sequence. Today’s Lab Today, you will isolate total DNA (genomic DNA and mitochondrial DNA) from salmon muscle tissue. A small section of the mitochondrial DNA molecule, called the COI gene, is what we will eventually sequence to help us identify the identity of the salmon species. But first we have to purify DNA from the salmon tissue away from all of the other macromolecules and ions that make up cells (proteins, lipids, carbohydrates, etc.) in order for the remaining experimental techniques to work. In future labs, we will make copies of the COI gene from our purified DNA using a technique called PCR, then use that amplified DNA in two separate techniques that allow us to determine the sequence of its nucleotides (A, T, G, C). Part 1: Observation of Salmon Sample Look carefully at your salmon samples with your naked eye. Compare them to those of other people around you who have the same species – do you note any differences in color or other appearance? If so, that might indicate that one of those samples was mislabeled at the time of purchase. Fill these notes into your notebook as well as the “Other Information, Notes, and/or Observations” section on Page 5 of the Project Overview handout and into the class excel spreadsheet. Part 2: Salmon DNA Isolation Protocol We will be using the “DNeasy” DNA Isolation Kit from Qiagen Laboratories to isolate genomic and mitochondrial DNA from your salmon samples. You and your lab partner(s) will each be doing your own DNA extraction. 1. Prepare your Experiment: a. Put on a lab coat and gloves and wipe your bench with water, then alcohol. Constant vigilance, seriously! b. You will each be assigned a unique sample code for each salmon sample (e.g. B####). When you have time between steps later during lab, add your samples’ information into the class excel spreadsheet. c. If you and your partner(s) will be sharing the same salmon sample for your DNA extraction, each separate extraction will be designated with a lowercase letter after your sample code (e.g. if your salmon sample code is B1003, the DNA extraction you perform 2 TBIOL 130, Winter 2023 Catching Cheaters I – Salmon DNA Extraction on that sample will be labeled B1003a, while your lab partner’s will be labeled B1003b). d. Using a sharpie, label the items used in this protocol according to Table 1 below (each student). Do not use labeling tape on the microcentrifuge tubes as this will throw off the weight of your tube during the centrifugation steps. Note that it is always safer to label both top and sides, in case the label should wear off from handling. Table 1: Labeled lab materials for salmon DNA extraction lab protocol. Each student should label the following items using the unique sample codes provided by the instructor. Label Item(s) Your salmon sample code ● Small petri dish (for cutting your sample). ● Original 1.5 mL microcentrifuge tube w/ your salmon sample (or a new tube if you had used a different container). ● Spin column w/ 2 mL collection tube. ● 1.5 mL microcentrifuge tube (+ “Lysis” label). ● 1.5 mL microcentrifuge tube (+ “DNA” label). ● Spin column w/ 2 mL collection tube. ● 1.5 mL microcentrifuge tube (+ “Lysis” label). ● 1.5 mL microcentrifuge tube (+ “DNA” label). (e.g. “B1003”) Your salmon sample code w/ extraction letter ‘a’ (e.g. “B1003a”) Your partner’s salmon sample code w/ extraction letter ‘b’ (e.g. “B1005b”) Figure 1 (on right): Labeled tubes and spin columns in preparation for salmon DNA isolation. The tubes in the top row are the DNeasy spin columns in collection tubes. On the bottom row are the microcentrifuge tubes. NOTE: Write your labels directly on the tube lids. Your labels will have different letter/numbers. e. Arrange your tubes in your tube rack/holder so that all items with the same sample codes are grouped together. You will also need the following reagents/buffers: ATL, K (proteinase K), AW1, AW2, AL, EtOH (ethanol), and AE. 2. Prepare your Salmon Samples: a. Cut out two small samples of salmon muscle tissue (the size of a matchstick head, or about this size. Use the labeled petri dish for that sample as a cutting surface. Use a NEW razor blade for each separate sample to avoid contamination. Try to gouge out a bit of muscle from the middle that does not have any gray tissue (fat) or is not dried out. 3 TBIOL 130, Winter 2023 Catching Cheaters I – Salmon DNA Extraction Check with your instructor before you proceed! This is a case where more is NOT better. It is actually better to err on the smaller side – too big a piece can generate too much DNA, gumming up the purification process. b. Once you have two pieces of tissue that are the right size, add one tissue piece to the “Lysis” tube matching your sample code (e.g. “B1003a”). Give your other sample piece to your partner in exchange for a piece from your partner’s salmon sample. Place your partner’s sample piece into your other “Lysis” tube matching their sample code (e.g. “B1005b”). Be sure that the salmon sample codes match that of the salmon samples you are using! c. Return the remaining salmon sample to your original labeled microcentrifuge tube, to be saved for confirmation of your results if you catch a cheater. Place it in the zip-lock bag in the back for storage at -20C. d. Add 180 μL of buffer ATL to each of the “Lysis” tubes with the cut tissue. e. Add 20 μL of Proteinase K to each “Lysis” tube. f. Mix thoroughly by vortexing for about 5 seconds. Repeat for a total of 2X vortex spins. g. Incubate the sample in the water bath (set to 56C) for 1 hour. During this hour-long incubation, shake or vortex the samples every 5-10 minutes or so. Purpose: Buffer ATL is required for tissue homogenization and cell lysis. It contains a mild detergent to help with this. Buffer also has a high concentration of salt, which helps DNA bind to the silica membrane in the Spin Filter. Proteinase K is an enzyme that degrades proteins, including those that help hold cells together within tissues. Degrading these proteins makes it easier to lyse cells, releasing all soluble macromolecules (including DNA) into the solution. h. During the incubation period, prepare your next buffer by adding 600 μL of ethanol (EtOH) to the tube labeled AL (this tube should already have 600 μL of buffer AL in it). Mix by pipetting up and down a few times. Relabel this tube as “AL + Et.” i. After the incubation with proteinase K is complete, remove each “Lysis” tube from the water bath and vortex at maximum speed for 5 seconds. Repeat for a total of 3X vortex spins. j. Add 400 μL of buffer AL+Et that you mixed up in step 2h to each of your “Lysis” tubes. Mix immediately by shaking the tube up and down, then vortex for 5 seconds. Repeat for a total of 3X vortex spins. Discard tube “AL+Et” into the tip waste container. Purpose: AL+Et buffer contains ethanol and salts, both of which allow DNA to bind to the silica membrane in the spin column. 3. Bind DNA to the Spin Column: 4 TBIOL 130, Winter 2023 Catching Cheaters I – Salmon DNA Extraction a. Pipet the mixtures from the “Lysis” tubes in step 2j (including any precipitate that may form) onto the corresponding labeled DNeasy Mini Spin columns that have already been placed into 2 mL collection tubes. Do not puncture the membrane of the spin column. Once you have pipetted everything out of the “Lysis” tubes, you can discard it in your waste container. b. Centrifuge the spin columns (with their collection tubes) at 8000 rpm for 1 min at room temperature. Make sure that the tubes rotate freely in the centrifuge without rubbing, and make sure the centrifuge is balanced. This can be done by placing two samples opposite from one another. Place the inner lid on the rotor before closing the outer lid and starting the centrifuge. c. Discard the flow-through liquid from the collection tubes into a liquid waste beaker and replace the columns back into the same collection tubes. Purpose: The flow through contains non-DNA molecules and ions. The DNA should be stuck to the silica membrane in the spin column. 4. “Wash” the DNA Attached to the Spin Column: a. Add 500 μL of buffer AW1 onto the spin columns, being careful not to puncture the membrane. b. Centrifuge the spin column at 8000 rpm for 1 min at room temperature. Again, recall that tubes MUST be balanced inside the rotor and that the inner lid should be in place before starting your centrifugation. c. Discard the flow-through liquid from the collection tubes and replace the column back into the same collection tube d. Add 500 μL of buffer AW2 onto the column (be careful not to puncture the membrane). Purpose: Buffers AW1 and AW2 are ethanol-based wash solutions used to clean the DNA that is bound to the silica membrane of the Spin Column. They remove residual salts, cellular debris, and proteins while allowing the DNA to stay bound to the membrane. e. Centrifuge the spin columns at 14,000 rpm for 3 min at room temperature (note the different speed and time. Remember to balance tubes and close inner lid!). f. Carefully transfer the spin columns into clean 1.5 mL microcentrifuge tubes labeled “DNA” with their corresponding sample codes (from step 1d). Make sure that the columns do not come into contact with the flow-through, since this will result in carryover of the wash buffers and possible contamination. g. Discard the collection tube and the flow-through liquid contained in it. 5 TBIOL 130, Winter 2023 Catching Cheaters I – Salmon DNA Extraction Purpose: The flow-through contains more non-DNA material (especially ions) washed away by buffer AW2. 5. Elute the DNA off the Spin Column: a. Add 200 μL of Buffer AE to the center of the white filter membrane in each spin column, without jabbing your tip into the membrane. Just drop down onto the membrane from above, centering to make sure that the entire membrane is wet. Purpose: Buffer AE is an elution buffer. It is composed mostly of water with a very low concentration of buffer. Placing it in the center of the small white filter membrane will ensure that the entire membrane is wetted. This will result in a more efficient and complete release of DNA from the silica spin column membrane. b. Let these tubes sit at room temperature for about 1 min. c. Centrifuge at 8000 rpm for 1 min at room temperature. Purpose: Centrifugation forces buffer AE through the silica membrane in the spin column. When buffer AE passes through the silica membrane, DNA that was bound in the presence of high salt is now selectively released by buffer AE which lacks salt. d. Discard just the spin column(s) in the tip waste jar. KEEP this last collection tube – this is your purified DNA! 6. Check the concentration of DNA in your samples (this may be done in the next lab period if you’re running out of time). a. When you are ready, notify your instructor that you are ready to measure the concentration of DNA in your samples. They will help you use the nanodrop to do this. Record your DNA concentration on the worksheet provided. b. Your purified DNA sample in the microfuge tube labeled “DNA” is ready to be stored until further steps. Place your purified DNA in the rack at the front of the lab, in numerical order. Your DNA will be stored at – 20°C until the next lab period. 7. Cleaning Up: 6 TBIOL 130, Winter 2023 Catching Cheaters I – Salmon DNA Extraction a. If you haven’t already done so… ● Add your samples’ information/data into the class excel spreadsheet. ● Place your purified DNA (in tube labeled “DNA”) in the designated tube rack in order based on number. Your DNA will be stored at -20°C until the next lab period. ● Return your remaining salmon sample to your original labeled microcentrifuge tube and place it in the zip-lock bag in the back. These samples will be stored in the freezer to go back to in the event that your sample was a cheater and we need to confirm those results before we go around pointing fingers at anyone. b. Empty your tip waste jar into the larger garbage can; rinse with water then with alcohol. c. Return all reagents and lab equipment (e.g. micropipettes, tips, racks, etc.). d. Rinse your scalpels or razor blades and place them on a paper towel to dry. e. Wipe your bench with water, then alcohol. f. Wash your hands before leaving lab – ALWAYS!!! 7 TBIOL 130, Winter 2023 Catching Cheaters I – Salmon DNA Extraction Lab Write-Up: Salmon DNA Extraction Please discuss these questions with your partner(s) but answer them INDIVIDUALLY. Short answer explanations need to be in your own words so I know that you understand your answers. For your lab write-up, type your final answers (using complete sentences) into a separate document. 1. Record the codes of your two salmon samples that you extracted DNA from. Complete the table below with the DNA concentrations of each sample. Include a table caption (located above the table) summarizing the information in this table when you submit your lab writeup. Sample #1 (Your sample “a”) Sample #2 (Your partner’s sample “b”) Official Sample Code Final DNA concentration (w/ units!) 2. Did you fill in the information on your salmon samples in the class spreadsheet? If your answer is “no,” go fill in your information now. 3. Of the 4 types of biological macromolecules, which would you expect to be most abundant in salmon muscle tissue? What type of biological macromolecule would be least abundant? Please explain your answer briefly in 2-3 sentences. 4. Briefly list the ingredients in the following solutions: i. ATL: ii. AE: iii. AW1 and AW2: 5. Which of the solutions from question 4 would be the BEST solvent for DNA? Briefly explain why in 2-3 sentences. Hint: Consider our brief discussion of polarity at the beginning of class when you answer this question. 6. The DNA extraction protocol removes soluble molecules other than DNA from solution. How were these soluble components (e.g. ions, other molecules, etc.) separated from DNA? Please explain briefly in 1-2 sentences. 8 TBIOL 130, Winter 2023 Catching Cheaters I – Salmon DNA Extraction 7. Based on your answers about the reagents in questions 4-6, why did DNA bind to the silica membrane in the spin column filter in steps 3 & 4, but flowed through into the collection tube in step 5? Please answer in 2-3 sentences. Hint: read carefully through the protocol! 9 TBIOL 130, Winter 2023 Catching Cheaters I – Salmon DNA Extraction Group Members: _____________________________________________ Lab Section: _______ Salmon Sample Information Sheet Record all of the relevant information on your samples. We will enter these data into the master spreadsheet. Your grade on this lab will be based in part on faithful record keeping. Sample #1 UNIQUE Code Number (provided by your instructor during lab. This is the label you will write on your tube during the isolation) Species Common Name Name Provided by Store (common name on label or display) Species Scientific Name (see table) Description from Store (wild or farmed, location of origin, fresh or frozen, cooked, etc.) Store or Restaurant Name and Address Date of Purchase or Date Sample was Obtained Store Personnel you talked to (if any) Other Information from Label (SKU codes, bill of lading, etc.) Other Information, Notes, and/or Observations 10 TBIOL 130, Winter 2023 Sample #2 Catching Cheaters I – Salmon DNA Extraction 11 TBIOL 130, Winter 2023 Catching Cheaters II – PCR Amplification CATCHING CHEATERS Part 2: PCR Amplification Lab Goals: 1. To understand how PCR amplification works and how it can be used to study DNA at the molecular level. 2. To set up/prepare your extracted salmon DNA for PCR for use in genotyping. Lab Preparation: 1. Read this lab protocol, with attention to the background on PCR and Central Dogma. 2. View the following PCR animations: ● See the DNA Learning Center tutorial: http://dnalc.org/resources/animations/pcr.html ● See the DNA Learning Center video: http://www.youtube.com/watch?v=2KoLnIwoZKU&list=PLD0D5F297744AD515&index=1 Introduction By now, you should all be familiar with proteins and enzymes, the biological macromolecules that control the almost infinite number of interactions and life processes in cells and living organisms. However, the molecular information that encodes these important proteins and enzymes is contained within the genome, the sequences of deoxyribonucleic acid or DNA that make up the blueprint of life found within each living organism. With amazing precision, this blueprint gets passed on from generation to generation of each species. The DNA molecule is a long polymer consisting of four different nucleotides. Each nucleotide is composed of a deoxyribose sugar, a phosphate group, and a nitrogenous base (Adenine, Guanine, Tyrosine, or Cytosine). Nucleotide chains of DNA are formed when the phosphate group of one nucleotide is joined to the deoxyribose sugar of the next nucleotide, creating the sugar-phosphate “backbone” of the DNA molecule. The various combinations of the four nucleotide bases protruding from the backbone make up the unique DNA code or sequence. Polymerase Chain Reaction (PCR) The Polymerase Chain Reaction (PCR) is a laboratory technique that allows researchers to amplify (make copies of) a specific DNA sequence of interest. PCR produces large amounts of a specific piece of DNA using only trace amounts of original (template) DNA. The portion of DNA we will amplify is a 648 base-pair region in the mitochondrial cytochrome c oxidase 1 gene (“CO1”), the standard gene that is used in the Barcode of Life project that identifies species using 1 TBIOL 130, Winter 2023 Catching Cheaters II – PCR Amplification “DNA barcoding”. This CO1 mitochondrial DNA sequence is commonly used to generate phylogenetic trees for a variety of organisms. One of the main reasons PCR is such a powerful tool is its simplicity and specificity. PCR requires the enzyme DNA polymerase to do the copying, two DNA primers to target the sequence of interest, a trace amount of the template DNA, and the four types of deoxyribonucleotide triphosphates (dNTPs) – adenine, guanine, thymine, and cytosine– that are the building blocks for DNA. DNA polymerase is an enzyme that uses the dNTPs to synthesize the complementary strand for a single-stranded DNA template from your organism of interest. In our case, the DNA template strand was isolated from your salmon tissue samples. However, DNA polymerase cannot start a new strand of DNA from scratch. In order to copy DNA, DNA polymerase needs a primer, which is a short strand of nucleotides that binds to the single-stranded DNA template, so that DNA polymerase can extend (or add onto) one end to make into a longer strand. The primers for PCR are specifically designed to bind and flank the DNA sequence of interest, thus ensuring that only the region of interest (and not the entire genome) is copied. How does PCR work? PCR involves a repetitive series of heating and cooling cycles, each of which consists of DNA template denaturation, primer annealing (binding), and extension of the annealed primer by DNA polymerase. This process is illustrated in Figure 1 with animations available online. Please view the following video animation by the DNA Learning Center on how PCR works: https://www.dnalc.org/view/15475-the-cycles-of-the-polymerase-chain-reaction-pcr-3d-animation.html 2 TBIOL 130, Winter 2023 Catching Cheaters II – PCR Amplification Figure 1: Illustration of the Polymerase Chain Reaction (from Pierce, B. Genetics: A Conceptual Approach 3rd ed.). The original double-stranded DNA template is first heated up to ~95C to separate into two singlestrands (denaturation). The solution is then cooled to ~55C so that the primers can bind (i.e. anneal) to its specific complementary regions on each single-stranded DNA template. Once the primers are annealed, the solution is then heated again to ~72C so that DNA polymerase can extend the primer and copy the single-stranded DNA template by adding deoxynucleotides to the 3’ end of the primer (in a single direction). These three steps—denaturation, annealing, and extension—comprise a complete thermal cycle. By repeating this process (~30-40 cycles), one can then make additional copies of the target DNA sequence. After each thermal cycle, the number of DNA template strands doubles, resulting in an exponential increase in the number of DNA template strands. After 40 thermal cycles, there will be 1.1 x 1012 more copies of the original number of template DNA molecules!! The DNA polymerase used in PCR must be thermally stable due to the high temperatures of denaturation. Thus, we use Taq polymerase for PCR, a DNA polymerase from a thermophilic bacterium (Thermus aquaticus) that lives in high-temperature hot springs. One of the amazing things about PCR is its specificity. PCR generates DNA of a precise length and sequence. On the first cycle, the two primers anneal to the original template DNA strands at opposite ends at the sequence of interest and on opposite strands. After the first complete thermal cycle, two new strands are generated that are shorter than the original template strands but still longer than the length of the DNA sequence of interest. It isn’t until the third thermal cycle that fragments of the precise length are generated (see figs. from textbook). What are we looking for in our salmon DNA? The gene we will amplify is a portion of the mitochondrial DNA plasmid. All eukaryotic cells have mitochondria, the small intracellular factories that carry out respiration. Mitochondria have their own DNA genome, which codes for several enzymes that are required for respiration. One of these is cytochrome-c oxidase, or COI. Mitochondrial DNA (mtDNA) has a faster mutation rate than genomic DNA. As a result, mtDNA genes accumulate mutations relatively rapidly over time, and therefore, closely related species have similar COI sequences, while less closely related species have COI sequences that are less similar (between two different genera such as Salmo and Oncorhynchus, a much lower percentage of the DNA bases would be the same). We will be using primers that correspond to the CO1 gene used in the Barcode of Life project. We ordered the Fish COI primers that are designed to amplify COI genes from fish, but not from other animals (including humans!). This will help to prevent contamination from human DNA from ruining our experiment. 3 TBIOL 130, Winter 2023 Catching Cheaters II – PCR Amplification Today’s Lab: In this week’s lab, we will set up our PCR reactions, and the PCR will be run for you after class (this takes ~3-4 hours). You will then perform gel electrophoresis on your PCR products in a few weeks to analyze whether our PCR reactions have worked. The best PCR products will then be sent off for sequencing. Our sequencing results will then be used to generate a phylogenetic tree to verify the species of your salmon sample and identify any market substitution and/or mislabeling of salmon. Lab Protocol – PCR Setup: We will dilute the DNA that you isolated from your salmon sample, pipette a small amount into a PCR tube, and add to it a PCR “master mix”. The master mix contains fish-specific COI primers to amplify part of the mitochondrial cytochrome-c oxidase gene as well as DNA (Taq) polymerase, deoxynucleotides (dNTPs), and buffer for optimal salt & pH conditions. The DNA is diluted to reduce the chance that contaminants (e.g. salts, polysaccharides or proteins) not fully washed away during DNA isolation may inhibit the reaction and PCR will not work. Your PCR tubes will then be placed into a thermal cycler and will undergo 40 cycles of amplification. After the PCR had completed, your tube will be held for you until a subsequent lab, where you will use gel electrophoresis to check your PCR product before sending it off for sequencing. WEAR GLOVES while setting up your PCR reaction! 1. Label a clean1.5 ml microcentrifuge for each sample with the appropriate Sample ID/code from last week. 2. If you have not yet done so last week, measure the concentration of DNA in your samples using the nanodrop with help from your instructor. Write the DNA concentration on your sample tube. 3. Dilute your DNA according to the following table: If your salmon DNA concentration is… Add to your 1.5 ml microcentrifuge tube… > 20 ng/μl 995 μl sterile water + 5 μl salmon DNA between 10-20 ng/μl 990 μl sterile water + 10 μl salmon DNA between 5-10 ng/μl 980 μl sterile water + 20 μl salmon DNA < 5 ng/μl 475 μl sterile water + 25 μl salmon DNA 4 TBIOL 130, Winter 2023 Catching Cheaters II – PCR Amplification 4. Close the lid and tap (“flick”) the base of the tube to mix. Return your non-diluted salmon DNA to the class box for storage. 5. Obtain a strip of thin-walled PCR tubes. You and your partner will share a strip of these tubes. Label each (tiny!) tube on the side with a sample ID number/code. The location of this label is important! If you label the lid, the ink may come off due to the hot thermal cycler lid. 6. Transfer 45 μl of each diluted DNA sample (from step 3) into the bottom of the appropriate labeled PCR tube. 7. Add 25 μl of the prepared “Master Mix” into each of your PCR tubes with sample DNA. Cap the PCR tubes tightly and tap/flick gently to mix. Tap tubes on the bench to get all the liquid back to the bottom of your tubes. Your resulting mixture should be green. 8. Bring your strip of tubes to the thermal cycler and place it into a row of empty reaction wells. The thermal cycler will hold the samples at 4˚ C until all groups have their samples ready. Your instructor will have positive and negative controls set up for the class, which will be run alongside the class samples. The reactions will take ~3 hours and stored at 4˚ C until we are ready to analyze them in a subsequent lab. Clean Up 1. Discard all used tips and tubes into the tip waste beaker. 2. Wipe down your bench with paper towels and detergent. 3. Used gloves and paper towels are disposed in TRASH. 4. Wash your hands before leaving lab! 5 TBIOL 130, Winter 2023 Catching Cheaters II – PCR Amplification Lab Write-Up: PCR Amplification Please discuss these questions with your partner(s) but answer them INDIVIDUALLY. Short answer explanations need to be in your own words so I know that you understand your answers. For your lab write-up, type your final answers (using complete sentences) into a separate document. 1. Complete the table below using the DNA concentrations of each sample from your group. Include a table caption (located above the table) summarizing the information in this table when you submit your lab write-up. Sample #1 Sample #2 Official Sample Code DNA Purity (A260/A280 Ratio) Final DNA Concentration (include units!) DNA Dilution (fill in amounts used w/ units) Sterile Water: Sterile Water: Salmon DNA: Salmon DNA: Discussion Questions: 1. We used Taq DNA polymerase to amplify the COI gene from your salmon DNA samples using PCR. Why was Taq specifically chosen as the enzyme for PCR amplification (as opposed to a eukaryotic source of DNA polymerase)? 2. Use the space below to draw a large linear segment to represent your salmon DNA sample. Draw the first 3 cycles of DNA amplification (during PCR) representing all the details of the cycles used in OUR EXPERIMENT (Lab 5). Drawings must be done by hand. Use colored pens or pencils if needed to distinguish template DNA, primers, and newly synthesized DNA. 3. Calculate the number of copies of your COI gene you’d amplify after… a. 8 PCR Cycles: b. 25 PCR Cycles: 6 TBIOL 130, Winter 2023 Catching Cheaters II – PCR Amplification c. 40 PCR Cycles: 4. What features of COI genes explain why this sequence was chosen (and not some other gene) to barcode animal species? 5. Answer the following about a positive control: a. Why is it important to include a positive control in each PCR run? b. What should we use for our positive control for this PCR run? c. If the positive control doesn’t produce a successful PCR product (and neither do any of the other DNA samples), then what can we conclude about the experiment? (What went wrong? What was defective?) 6. Answer the following about a negative control: a. Why is it important to include a negative control in each PCR run? b. What should we use for our negative control for this PCR run? c. If we find that DNA was amplified in the negative control reaction, what would you conclude about the PCR run overall? 7. List at least 3 major differences between DNA replication and Polymerase Chain Reaction (PCR). 7 TBIOL 130, Winter 2023 Catching Cheaters III – Agarose Gel Electrophoresis CATCHING CHEATERS Part 3: Agarose Gel Electrophoresis Lab Goals ● ● ● ● To analyze your PCR results using DNA gel electrophoresis. To explain the principles of PCR amplification, gel electrophoresis, & DNA sequencing. To practice loading, running, and analyzing a DNA agarose gel. To apply DNA sequence analysis to an environmental issue. Lab Preparation & Study Questions 1. Read this lab, with attention to the background on PCR and gel electrophoresis. 2. View the following tutorials and video animations from the DNA Learning Center: ● PCR tutorial: http://dnalc.org/resources/animations/pcr.html ● PCR animation video: https://youtu.be/2KoLnIwoZKU ● Gel electrophoresis video: http://dnalc.org/resources/animations/gelelectrophoresis.html ● DNA sequencing video: https://youtu.be/6ldtdWjDwes 3. What is the purpose of PCR? How does PCR work? What reagents are required? What happens at each temperature? What is this a “chain reaction”? 4. In gel electrophoresis, why does the DNA migrate through the gel towards the positive pole? 5. Sketch what you would expect to see IF you ran a gel with your PCR sample. Hint: What is the size of the full-length COI PCR product? Draw in the bands on the gel. 6. If your gel electrophoresis is successful, what will you be sending out for sequencing at the end of this lab? Introduction In today’s lab, we will use gel electrophoresis to finally see whether or not our PCR (from our previous Catching Cheaters lab) worked and we were able to amplify a section of the Salmon COI gene. The gel will also allow us to assess the quantity and quality of the DNA template (isolated in our first Catching Cheaters lab), so that we can use this information to know which samples to send out for sequencing. 1 TBIOL130: Intro Bio II (Winter 2023) Catching Cheaters III – Agarose Gel Electrophoresis REVIEW: Polymerase Chain Reaction (PCR) Polymerase Chain Reaction (PCR) is a laboratory technique that allows researchers to amplify (make lots of copies of) a specific DNA sequence of interest in a test tube (in vitro). PCR produces large amounts of a specific piece of DNA using only trace amounts of original (template) DNA. Starting with DNA from a drop of blood, a hair follicle, or a cheek cell, it is possible to generate billions of copies of a specific DNA fragment. Before PCR, many molecular biology techniques were labor intensive, time consuming, and required a high level of technical expertise. Working with trace amounts of DNA also made it difficult for researchers in biological fields such as pathology, zoology, or ecology to incorporate DNA analysis into their research. PCR had an impact on four main areas of biotechnology: gene mapping, molecular cloning, DNA sequencing, and gene detection. PCR technology streamlined these aspects of molecular biology, making it one of the most accessible and widely used tools in genetic and medical research. PCR is now used as a medical diagnostic tool to detect specific mutations that may cause genetic disease, in legal investigations to identify or eliminate suspects using molecular genetics, and in sequencing the human genome. One of the main reasons PCR is such a powerful tool is its simplicity and specificity. PCR requires the enzyme DNA polymerase (Taq) to do the copying, two DNA primers to target the sequence of interest, a trace amount of the template DNA, and the four types of deoxyribonucleotide triphosphates or dNTPs ( dATP, dCTP, dGTP, and dTTP) that are the building blocks for DNA. How does PCR work? PCR involves a repetitive series of heating and cooling cycles, each of which consists of DNA template denaturation, primer annealing (binding), and extension of the annealed primer by DNA polymerase. NOTE: The DNA polymerase used in PCR must be thermally stable due to the high temperatures of denaturation, thus we use Taq polymerase from a thermophilic bacterium (Thermus aquaticus) that lives in high-temperature hot springs for PCR. This process is illustrated in Figure 1 with video animations from the DNA Learning Center – please see links on Canvas and on the first page of this lab. The original double-stranded DNA template is first heated up to ~95C to separate into two singlestrands (denaturation). The solution is then cooled to ~55C so that the primers can bind (i.e., anneal) to its specific complementary regions on each single-stranded DNA template. Once the primers are annealed, the solution is then heated again to ~72C so that DNA polymerase can extend the primer and copy the single-stranded DNA template by adding deoxynucleotides to the 3’ end of the primer (in a single direction). 2 TBIOL130: Intro Bio II (Winter 2023) Catching Cheaters III – Agarose Gel Electrophoresis Figure 1: Illustration of the Polymerase Chain Reaction (from Pierce, B. Genetics: A Conceptual Approach 3rd ed.). These three steps—denaturation, annealing, and extension—comprise a complete thermal cycle. By repeating this process (~30-40 cycles), one can then make additional copies of the target DNA sequence. After each thermal cycle, the number of DNA template strands doubles, resulting in an exponential increase in the number of DNA template strands. After 40 thermal cycles, there will be 1.1 x 1012 more copies of the original number of template DNA molecules!! For our lab, the portion of DNA we amplified is a 648 base-pair region in the mitochondrial cytochrome c oxidase 1 gene (“CO1”), the standard gene that is used in the Barcode of Life project that identifies species using “DNA barcoding”. This CO1 mitochondrial DNA sequence is commonly used to generate phylogenetic trees for a variety of organisms. Agarose Gel Electrophoresis To determine whether our PCR reaction from our previous lab was successful, you will visualize and assess the size and quality of the PCR product, the fragment of DNA corresponding to the CO1 gene, using gel electrophoresis. Gel electrophoresis separates DNA fragments according to their relative sizes (number of base pairs (bp)). This is done by loading the DNA fragments into an agarose gel slab, which is placed into a chamber filled with a conductive buffer solution. A direct current is passed between wire electrodes at each end of the chamber. Since DNA fragments are negatively charged, they will be drawn toward the positive pole and repelled by the negative pole when placed in an electric field. 3 TBIOL130: Intro Bio II (Winter 2023) Catching Cheaters III – Agarose Gel Electrophoresis The matrix of agarose gel acts as a molecular sieve. The gel impedes the movement of the fragments; the more agarose in the gel, the slower the DNA moves through it. More importantly, smaller DNA fragments can move more easily than larger ones. Over a period of time, smaller fragments will travel farther than larger ones. Fragments of the same size stay together and migrate in what appears as a single “band” of DNA in the gel. Figure 2 shows an example of an agarose gel with stained DNA fragments separated by size. The bands in lane 1 are the molecular ladder which is a sample containing DNA fragments of known sizes. In this case, the molecular mass ruler contains 1,000 bp, 700 bp, 500 bp, 200 bp, and 100 bp fragments. The molecular ladder is used to determine the size of unknown DNA fragments and to track the position of expected fragments. Using the molecular ladder in lane 1 as a guide (see Figure 2), approximately what size is the band in lane 2? ____________ What size is the band in lane 3? ____________ Figure 2: Example of a gel after electrophoresis. The banding pattern of the molecular ladder is shown in column 1. Lanes 2-8 show DNA bands and fragments of varying sizes. If we were successful in amplifying the COI gene from our salmon samples using PCR, we should see a single clear band representing a DNA fragment of ~650 bp. If we had some crosscontamination, we will see multiple bands. And, of course, if the cells were not lysed effectively or the DNA was lost during one of the wash steps, we won’t see anything at all. Since DNA is colorless, the position of the DNA fragments is not visible in the gel unless the gel is stained. The loading dyes in the gel loading buffer have pigments that can be seen using normal white light. These help prevent us from running the gel too long (what would be the result if this happened?). It is important to realize that the location of the loading dyes does NOT reflect the positions of the DNA fragments. The gel contains a different dye that has positively charged ions that strongly binds to the DNA fragments. This dye fluoresces under blue light and makes the DNA bands within the gel become visible when the gel is placed on the blue light box. DNA Sequencing While using gel electrophoresis allows us to visualize the size of DNA segments copied during PCR, DNA sequencing allows us to determine the exact sequence of base pairs in our isolated DNA segment. Sequencing of DNA has produced an explosive growth of information. Sequencing was slow and difficult until the last few years, when automated sequencers have made it possible to read DNA sequences quickly and cheaply. The human genome, as well as complete genomes of several other organisms, has now been sequenced (much faster than predicted), partly due to the recent advances in DNA sequencing technology. We do not have a sequencing machine at UW Tacoma, but we can send our samples to biotechnology companies that specialize in sequencing methodologies. After checking whether or not our PCR was successful, we will 4 TBIOL130: Intro Bio II (Winter 2023) Catching Cheaters III – Agarose Gel Electrophoresis prepare our samples to be shipped to Eurofins MWG Operon (www.operon.com), a company that provides DNA sequencing services (in addition to other genomic services and products). Sequencing of DNA has produced an explosive growth of information. Sequencing was slow and difficult until the last few years, when automated sequencers have made it possible to read DNA sequences quickly and cheaply. The human genome, as well as complete genomes of several other organisms, have now been sequenced (much faster than predicted), partly due to the recent advances in technology. The modern sequencing reaction is based on an elegant method devised by Frederick Sanger, in 1975, for determining the sequence of DNA nucleotides. Just as in PCR, sequencing starts with a DNA template, deoxynucleotides, Taq polymerase, and buffer. Unlike PCR, the sequencing reaction uses only one primer, so that all new sequences are generated in one direction from only one of the DNA strands. Another critical difference is that, while PCR is often performed on genomic DNA or other mixed DNA with the primers acting to select the region that is copied, DNA sequencing is performed using a PCR product as the DNA template. In other words, the DNA template is a single gene (copied by PCR) rather than a mixed sample. In our case, we will run our sequencing reaction using the COI PCR product from the previous lab, which should consist of lots of copies of the COI gene (and nothing else!). The sequencing facility will add a reaction mixture that contains fluorescently labeled di-deoxynucleotides (ddNTPs) in addition to the normal deoxynucleotides (dNTPs) in the reaction mix. The template DNA is copied by Taq polymerase until it happens to add a ddNTP, which causes the copied DNA strand to terminate. As this process happens again and again in the reaction tube, eventually we generate a mixture of all different lengths of copies of the template DNA, each one ending with a ddNTP. When these fragments are separated by size, the sequencer can read the length and dye color of each fragment, and put this together to provide a print-out of the DNA sequence. The automated sequencing machines used at Eurofins MWG Operon can read at least 96 samples at one time, at a cost of a few dollars per sample. By using thin capillary tubes instead of flat electrophoresis gels like the ones we have used in our lab, more samples can be analyzed at one time, with minimal preparation time. The field continues to advance, with exponential increases in speed and cost-effectiveness. New sequencing techniques, referred to as “Next-gen” sequencing, have made the sequencing of entire genomes routine and at a reasonable cost. 5 TBIOL130: Intro Bio II (Winter 2023) Catching Cheaters III – Agarose Gel Electrophoresis Laboratory Protocol Part I. Agarose Gel Electrophoresis You will analyze your PCR product by running agarose gel electrophoresis. Electrophoresis separates DNA fragments according to their relative sizes (number of base pairs (bp)). This is an exciting moment, when you get to see whether or not your PCR reactions worked! WEAR GLOVES while working with your gels! 1. You will be sharing one agarose gel and one electrophoresis chamber with several other lab members. 2. Obtain your PCR reaction from the previous week. 3. Fill in you and your lab partners’ sample code in the table on page 10 (Group Data Sheet), with the order that you will load each sample to the gel. This step is VERY IMPORTANT! If you do not do it, you may get mixed up about which sample is in which lane. 4. When you are ready to begin loading your gel, ask your instructor to come demonstrate by loading the molecular ladder. Set your micropipette to a volume of 10 μL (which micropipettor do you use?). Using a clean tip for each sample, carefully load the samples into the wells of the gel by transferring 10 μL of your PCR product (with loading dye added) into the bottom of the wells. Follow the order and loading volume shown in the data table (and make note of where the positive and negative controls are located on the gel). Try not to get bubbles in the wells. After you’ve finished loading your gel, you may have a few empty lanes; this will not affect running the gel. ***DO NOT THROW AWAY YOUR PCR TUBE!!!*** If your PCR was successful, you will submit a sample of your PCR product for sequencing. *Tip: To avoid bubbles, push down ONLY until the FIRST stop of your micropipette for both drawing up your sample and loading your gel. (Remember, it’s the SECOND stop that pushes extra air out of the pipette tip.) It’s ok for a tiny amount to be left in your pipette tip – it’s better than having the entire sample float out of the well. If there are bubbles, you can “chase” them out with the tip of your pipet. 5. When all the samples have been loaded, place the lid onto the electrophoresis chamber, matching the electrodes black-to-black and red-to-red. Connect the electrical leads to the power supply, ensuring that the colors of the wire connectors match those on the power supply. 6. Turn on the power supply and ensure it is set for 200 V. Run the gel for 30-35 minutes. You should start to see little bubbles appear at the two electrode ends as current is applied. Even though you can’t see your DNA samples, you can track their progress by watching the dye front as it migrates towards the bottom of the gel. Make sure that your loading dye is moving towards the opposite end of the gel. 6 TBIOL130: Intro Bio II (Winter 2023) Catching Cheaters III – Agarose Gel Electrophoresis Note: Normally, you would need to add loading dye to your DNA prior to loading your gel. However, the PCR buffer that we used for this lab and the molecular ladder already contains loading dye, thus you will not need to add any additional dye to your samples. 7. When the gel has finished running (after ~30-35 minutes), turn off the power supply and remove the lid from the gel box. Carefully remove the tray containing your gel and bring it over to a gel box to visualize. Please carry it over a paper towel so that you do not drip buffer all over the lab! The gel is also slippery and can easily slide off! 8. You are now ready to image and make a record of your gel. We will be examining the gels using the Digi Doc-it system in the room. Gently nudge your gel off the gel tray and slide it onto the blue light box. Your instructor will help you to take an image of your gel, which you can view on the computer and save it as an image file. Your images will be uploaded onto Canvas for you to download and view/print out. 9. When you are finished, wipe down the light box with paper towels and discard the gel in the tray in the hood. Pour the electrophoresis buffer back into the bottle (to be reused). Rinse the gel tray and the gel box and leave inverted on paper towels to dry. Pipet tips and tubes may be discarded in the trash. Gel Analysis 1. Draw the bands on your gel on page 9, using the gel schematic shown. 2. Label the sizes of the bands in your molecular ladder (Lane 1) according to the DNA ladder (Figure 3). 3. Compare the lane with you and your lab partners’ PCR samples with the positive control lane using this DNA ladder as a size reference. How does your PCR band compare to the control? Is your PCR product the correct size? Did your PCR work? Fill in this info on the table on page 7. Be sure the information on the table matches the lanes of your gel. Figure 3: Low Range DNA Ladder (from Thermo Scientific) 7 TBIOL130: Intro Bio II (Winter 2023) Catching Cheaters III – Agarose Gel Electrophoresis II. Sequencing Reactions (Set-up only) After examining the gels of our PCR products, we can now send your samples out for sequencing. Your PCR samples will then be shipped overnight express to Eurofins MWG Operon for sequencing. We will receive our salmon COI gene sequences in a few weeks, which we will then use to analyze to identify our salmon samples and build a phylogenetic tree in a subsequent lab. The sequencing reaction itself will be performed by the Eurofins sequencing facility. All we need to do is to add labels with unique bar codes (provided by Eurofins) onto the PCR samples that we are sending out for sequencing, and record on our Google Sheet which bar code corresponds with which sample. It is critically important that our records are accurate so that we do not confuse our samples! We will then ship overnight these PCR samples along with an aliquot of our COI sequencing primer to Eurofins. The sequencing facility will purify your PCR product and add the sequencing reagents that contain your primer and the fluorescently labeled di-deoxy-nucleotides (ddNTPs). They will use a thermal cycler to make multiple copies of the template DNA, but unlike PCR, these copies will be of various lengths, and will contain the fluorescently labeled ddNTPs. When these copies are run in a gel, the sequencer can read the length and dye color of each fragment and put this together to provide a print-out (file) of the DNA sequence, which we will analyze in two weeks. Preparing Samples for Sequencing: 1. Obtain a clean 1.5 or 2 ml microcentrifuge tube; one for each PCR sample that worked based on your gel results. 2. Label the lid of the tube with your unique salmon code. 3. Transfer ALL of your remaining PCR product into the new labeled tube. 4. Place your labeled tube into the rack in front of class. 5. Your instructor will send these out to Eurofins for sequencing along with our COI sequencing primer. We should receive our sequences in time for our last lab of the quarter for us to analyze. 8 TBIOL130: Intro Bio II (Winter 2023) Catching Cheaters III – Agarose Gel Electrophoresis Lab Write-Up: Agarose Gel Electrophoresis Please discuss these questions with your partner(s) but answer them INDIVIDUALLY. Short answer explanations need to be in your own words so I know that you understand your answers. For your lab write-up, type your final answers (using complete sentences) into a separate document. Group Data Sheet: Agarose Gel Electrophoresis (Salmon COI Gene) GEL #: Lane # Sample (Code & Student Initials) Amount Loaded Estimated DNA fragment sizes? PCR worked? Various N/A 1 Low Range DNA Ladder 10 µL 2 Positive Ctrl 1 ( ) 10 µL 3 Positive Ctrl 2 ( ) 10 µL 4 Negative Ctrl 10 µL 5 6 7 8 9 10 11 12 13 14 15 9 TBIOL130: Intro Bio II (Winter 2023) Catching Cheaters III – Agarose Gel Electrophoresis ● Use the following schematic to draw the bands from your agarose gel below using your gel image (posted on Canvas) as a guide. ● Label the sizes of the bands in your molecular ladder (see Figure 3). You will also need to indicate whether the PCR reaction from each lane worked by indicating “+” or “-“ under each well. Answer the following questions: 1. What is the expected size of your PCR product (COI gene)? 2. Use your gel results to evaluate your positive and negative controls. Did your controls for this PCR work? Briefly explain how you know you can “trust” the results of your PCR using your positive and negative controls. 3. What was the Sample Code for your PCR product? What was the lane number on gel where you loaded your PCR product? 4. Briefly evaluate the results of your PCR. How do your PCR results compare to the positive and negative controls? Based on the size of your PCR product(s), did your PCR work? 5. If your PCR worked, briefly describe the next steps in your goal to identify the species of Salmon in your Catching Cheaters project. If your PCR did not work, briefly describe what happened (i.e. why didn’t your PCR work?) and what you could do to prevent this issue in future attempts. 10 TBIOL130: Intro Bio II (Winter 2023) Problem Set IV Mutagenesis TBIOL 130 (Winter ‘23) Name: Score: Answer the following questions to the best of your ability; this exercise is worth 30 points and must be uploaded to Canvas by midnight on Friday of Week 5 (Feb. 3rd). You may work together with a group to complete this exercise, but everyone must submit their completed problem sets separately. Please note that “work together with a group” does not mean that you can submit identical answers. In other words, you can consult with your peer(s) on the answer (or the means to come up with the answer) but you need to answer the questions in your own words. You may provide your answers in a separate file or within this worksheet (save as a .pdf file) but for the latter, remember to TYPE YOUR ANSWERS IN RED (or another color of your choice)! Part I: Manipulating DNA Sequences 1. (6 points) Below is the gene sequence for a short polypeptide (top strand is coding strand; wild-type). You have been provided with 6 mutated versions of this coding sequence, each producing different outcomes once the corresponding polypeptide is expressed. For each mutated sequence, provide the corresponding a) mRNA and b) polypeptide sequence. This information will be necessary for you to c) determine what kind of mutation was introduced into the gene (be specific!). • For full credit, you must provide the complete mRNA/polypeptide sequence and not just the portions that are affected by the mutation. Additionally, you need to include the appropriate direction markers (5’, 3’; N, C) in your answers! • For ease of grading, provide polypeptide sequence using the 3-letter amino acid code (e.g. His for Histidine or Gln for Glutamine). • The wild-type mRNA and polypeptide sequences were not provided but may be helpful to you as you determine which type of mutation was introduced in the mutated sequences. Feel free to use the space provided to fill in the wild-type sequences. DNA: 5’ – ATG GTC GGT TGT ATA AAG CCC AAT GAG TAA – 3’ 3’ – TAC CAG CCA ACA TAT TTC GGG TTA CTC ATT – 5’ mRNA: Polypeptide: a. Type of Mutation: Coding DNA: . 5’ – ATG GTC GGT TGT ATA AAG CCC AAT GGA GTA A – 3’ mRNA: Polypeptide: b. Type of Mutation: Coding DNA: mRNA: Polypeptide: . 5’ – ATG GTC GGT TGT ATA AAG CCC AAT GAC TAA – 3’ Problem Set IV Mutagenesis TBIOL 130 (Winter ‘23) c. Type of Mutation: Coding DNA: . 5’ – ATG GTT GGT TGT ATA AAG CCC AAT GAG TAA – 3’ mRNA: Polypeptide: d. Type of Mutation: Coding DNA: . 5’ – ATG GTC GGT TGT ATA AAG CCC AAG AGT AA – 3’ mRNA: Polypeptide: e. Type of Mutation: Coding DNA: . 5’ – ATG GTC GGT TGA ATA AAG CCC AAT GAG TAA – 3’ mRNA: Polypeptide: f. Type of Mutation: Coding DNA: . 5’ – ATG GTC GGT TGT ACA AAG CCC AAT GAG TAA – 3’ mRNA: Polypeptide: 2. In your research lab, you have recently discovered a mutant strain (mut205) of Arabidopsis thaliana (plant) that has a propensity to die off early in development (i.e. the mutations are lethal) and you have narrowed down the genetic cause to mutations in histone H4 (one of the subunits necessary for a histone protein). Below is an amino acid alignment (103 amino acids; single letter amino acid code) you generated comparing the amino acid sequence for histone H4 from wild-type and mut205 A. thaliana plant cells. • In the alignment, conserved positions (i.e. positions where there are no changes to the amino acid sequence) are indicated with “*” while differences in amino acid sequence are indicated with “.” or nothing. a. (1 point) What is the functional role of histones in eukaryotic cells? Problem Set IV Mutagenesis TBIOL 130 (Winter ‘23) b. (2 points) Examine the amino acid alignment and determine what kind of point mutations occurred in order to explain the changes in the amino acid sequence for mut205. c. (3 points) Considering the nature of the amino acid changes that you outlined in Question #2B, what effect would you expect these mutations to have on the function of histone H4 if they were located on the surface of the protein? d. (3 points) Considering the nature of the amino acid changes that you outlined in Question #2B, what effect would you expect these mutations to have on the function of histone H4 if they were located within the core internal structure of the protein? e. (4 points) For simplicity, we will assume that the amino acid changes shown in mut 205 are located on the surface of the histone H4 protein. Speculate why these specific amino acid changes are detrimental (lethal) early in development. Be specific with your answer. Part II: Mutagenesis in Action 3. Viral genomes (particularly those of RNA viruses) are subject to mutation, which gives rise to different variants (also called strains) of the virus within the population. You may have heard about the “UK strain” of SARS-CoV-2 (B.1.1.7), which was determined to be more contagious than the other circulating SARS-CoV-2 strains that we had seen during the COVID-19 pandemic. Well, it turns out that the B.1.1.7 strain of SARS-CoV-2 has accumulated over 20-point mutations that make it distinct from the other circulating strains. We will return to the discussion for how these specific changes alter the viral proteins when we discuss protein structure in a few weeks. For now, let’s think about mutations and their consequences. a. (2 points) What is a genome? How would you explain this concept to a layman without using biological terms like ‘genes’ or ‘genetic information’? Problem Set IV Mutagenesis TBIOL 130 (Winter ‘23) b. (3 points) Many of the significant mutations in the B.1.1.7 variant are located in the S gene of the virus, which encodes the viral surface protein that binds to the host-cell receptor and facilitates viral entry into the host cell. Would you expect to find circulating variants of SARS-CoV-2 that have loss-of function mutations in the S gene? Briefly explain your answer. c. (3 points) Given the role of the S gene product in the viral life cycle, would you expect to find missense or nonsense mutations (choose one) more frequently near the beginning of this gene in circulating viral variants? Explain your reasoning. d. (3 points) Given the role of the S gene product in the viral life cycle, would you expect to find missense mutations or frameshifts (choose one) more frequently in this gene in circulating viral variants? Explain your reasoning. BONUS (1 point): In addition to several mutations in the S gene, hypothesize where else in the SARS-CoV-2 genome we might expect to find mutations that make the virus more infectious. Be sure to briefly explain your gene choice as part of your answer. Use the viral genome summary figure shown below to see some notable viral protein candidates and use the following website to find more information about the function of the viral proteins. • https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7293463/ • Figure from https://www.ncbi.nlm.nih.gov/books/NBK554776/figure/article-52171.image.f5/
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