MICR 290 M08 Antibiotic Resistance Lab PDF

MICR 290 oiw ANTIBIOTIC RESISTANCE LAB MODULE 08 CONTROLLING BACTERIAL GROWTH Please note: This course was designed to be interacted and engaged with using the online modules. This Module Companion Guide is a resource created to complement the online slides. If there is a discrepancy between this guide and the online module, please refer to the module. How can you help protect the integrity and quality of your Queen’s University course? Do not distribute this Module Companion Guide to any students who are not enrolled in MICR 290 as it is a direct violation of the Academic Integrity Policy of Queen’s University. Students found in violation can face sanctions. For more information, please visit https://www.queensu.ca/academic- calendar/health-sciences/bhsc/. MODULE 08 COMPANION GUIDE MICR 290 TABLE OF CONTENTS SECTION 00: Module Overview............................................................................................................................ 3 SECTION 01: Sterilization, Disinfection, Antisepsis, and Sanitization.............................................................. 4 SECTION 02: Chemical Methods of Control......................................................................................................11 SECTION 03: Physical Methods of Control........................................................................................................18 SECTION 04: Testing Antibiotic Susceptibility..................................................................................................24 SECTION 05: Antibiotic Resistance in the Clinic...............................................................................................33 SECTION 06: Antibiotic Discovery......................................................................................................................39 ANTIBIOTIC RESISTANCE LAB|MICR 290 M08 PAGE 2 MODULE 08 COMPANION GUIDE MICR 290 SECTION 00: MODULE OVERVIEW Bacteria are ubiquitous, and the ability to control their growth is critical in certain contexts. In healthcare settings and applications, the growth of bacteria on medical devices must be controlled in order to minimize the risk of causing infection. To treat a bacterial infection, the growth of the pathogen in the body must be controlled. In food production and processing industries, bacteria can often be beneficial; however, controlling bacterial growth is extremely important in order to prevent the spoilage of food products and potential food-borne illnesses. Although this module focuses principally on the control of bacterial growth, many of the concepts you will learn are equally relevant to the control of other microorganisms such as viruses and fungi. Watch the video of your instructor, Dr. Lohans, introducing the concepts that are covered in Module 08 (1:55). Please refer to the module for this video. After completing this module, you will be able to: 1. Compare and contrast sterilants, disinfectants, antiseptics, sanitizers, and antibiotics, and explain how common physical and chemical methods are able to control bacterial growth. 2. Compare and contrast common techniques for measuring the antibiotic susceptibility of a bacterial strain. 3. Interpret the experimental results generated by common techniques used for testing antibiotic susceptibility, and compare them to published clinical breakpoints to determine the antibiotic susceptibility of a bacterial strain. 4. Interpret antibiograms in order to recommend and justify which antibiotic should be used to treat an infection caused by a specific bacterial species. 5. Describe the processes and challenges associated with the development of new antibiotics. Page Link: https://player.vimeo.com/video/465869511 End of Section 00 ANTIBIOTIC RESISTANCE LAB|MICR 290 M08 PAGE 3 MODULE 08 COMPANION GUIDE MICR 290 SECTION 01: STERILIZATION, DISINFECTION, ANTISEPSIS, AND SANITIZATION The extent to which bacterial growth must be controlled depends on the context. If a scalpel is being used for invasive surgery, it must be completely free of bacteria. On the other hand, the presence of bacteria on a blood pressure cuff does not represent a significant infection risk (assuming intact skin), and it is less important to remove all of the bacteria from the cuff. In this section, you will learn about general processes for controlling bacterial growth, and explore when they might be applied according to their relative levels of efficacy. Levels of Control for Bacterial Growth There are several defined levels of control for bacterial growth. These levels are distinguished based on how and where the bacterial growth is being controlled. Learn about the processes that are commonly used to control bacterial growth, from least to most rigorous. Sterilization Sterilization refers to a process that completely eliminates all bacteria. For example, you learned about autoclaving in Module 01, which is one of several physical techniques for sterilizing medical tools, equipment, and reagents in the lab. Chemicals known as sterilants can also be used to sterilize tools and equipment. Disinfection Disinfection refers to a process that kills most but not all bacteria that are present on a surface, tool, or piece of equipment. Some difficult-to-kill bacteria survive disinfection, and so disinfection cannot be used to sterilize surfaces or equipment. Chemicals used to disinfect objects are known as disinfectants. Antisepsis While disinfection involves killing bacteria on inert objects, antisepsis involves killing bacteria on living tissues. Chemicals that are used to kill bacteria on living tissues are known as antiseptics. Note that antiseptics do not sterilize the living tissue, and leave some living bacteria. Some of the chemicals used as antiseptics are also known as biocides or germicides (although these terms are often used more broadly). Germicides are bactericidal, meaning that they kill bacteria, but some antiseptics are bacteriostatic and inhibit the growth of bacteria without killing them. You have previously come across the term aseptic technique, which describes measures for preventing bacterial contamination. Asepsis refers to the absence of bacterial contamination, while the related term sepsis refers to a life-threatening infection commonly caused by bacteria. Sanitization Sanitizers are less lethal to bacteria than disinfectants, antiseptics, and sterilants. However, sanitizers are generally safer for skin, and are often used to clean equipment and facilities that are often contacted by humans. The process of using a sanitizer is known as sanitization. ANTIBIOTIC RESISTANCE LAB|MICR 290 M08 PAGE 4 MODULE 08 COMPANION GUIDE MICR 290 You may have noticed some conceptual overlap between antibiotics and sterilants, disinfectants, antiseptics, and sanitizers. It is important to note that the distinction comes down to the selectivity of these agents in killing bacteria. As you can likely now appreciate, most antibiotics are extremely selective in their level of control, and inhibit or kill bacteria by targeting specific pathways. By contrast, sterilants, disinfectants, antiseptics, and sanitizers tend to kill bacteria by more general mechanisms that target the major components found in all cells. For example, some of these agents: increase the permeability of the bacterial membrane, causing leakage of the contents out of the cell. non-specifically damage proteins, chemically modifying the side chains of amino acids, and/or causing proteins to aggregate and unfold. modify the chemical structure of nucleic acids, interfering with DNA replication, transcription, and translation. These major components are all essential for bacterial growth and replication, and targeting them kills bacterial cells (or, at least, inhibits their growth). Infographic comparing different agents of bacterial control by their level of selectivity. Use what you have learned about antibiotics and other agents (e.g. sterilants, disinfectants, antiseptics, and sanitizers) to answer the questions. 1 of 2: If sterilization, disinfection, antisepsis, and sanitization all utilize similar methods to kill bacteria, why are some more effective than others? Feedback: Dr. Lohans’ Response: ANTIBIOTIC RESISTANCE LAB|MICR 290 M08 PAGE 5 MODULE 08 COMPANION GUIDE MICR 290 Some methods are more effective at killing bacteria than others because of how potently they interact with their targets. For example, sterilants that target proteins are extremely reactive molecules that very rapidly modify and inactivate proteins. While some disinfectants and antiseptics also target proteins, they are far less reactive molecules, and modify their targets more slowly. There are similar considerations for physical methods: heating a sample may kill some bacteria, while the more extreme measure of heating a sample at high pressure can kill all bacteria. There is a general trade-off: stronger methods of control kill bacteria more efficiently, but are more likely to harm human cells. 2 of 2: Why might the general mechanisms of control you have learned about in this module have a greater impact on human cells than antibiotics? Feedback: Dr. Lohans’ Response: Sterilants, disinfectants, antiseptics, and sanitizers kill bacteria by general mechanisms that can also target human cells. For example, many of these agents target all proteins. As proteins play a vital role in all living cells, these agents are more likely to have toxic effects on human cells. The antiseptics and sanitizers used on humans tend to applied on the skin and the mouth, and have relatively limited exposure times. In contrast, most antibiotics are not very toxic, and can be administered into the body (e.g. intravenously, orally, intramuscularly) so that they can reach the site of a bacterial infection. The remainder of this section will discuss methods of testing the efficacy of sterilization, as well as the different levels of disinfection. Testing the Efficacy of Sterilization The proper sterilization of a piece of equipment or tool requires that all bacteria have been killed. It is essential to confirm that the sterilization process has worked properly, as incomplete sterilization could have life-threatening consequences (e.g. infections resulting from surgical implements contaminated with bacteria). As such, there must be controls in place to test whether or not sterilization is working properly. The efficacy of sterilization can be tested using biological indicators and chemical indicators. As you learned previously, autoclaves kills bacteria through the use of high pressure steam. In this section, you will focus on the use of indicators to confirm that an autoclave is working properly. Related indicators can also be used to test other sterilization methods. Biological Indicators Biological indicators are typically living bacterial cells which are used to test the efficacy of a sterilization method. However, not all bacteria are the same in terms of how easy they are to kill. Compared to vegetative bacteria, that is bacteria that are actively growing and replicating, bacterial spores are very difficult to kill. Bacterial spores are a dormant form of bacteria that are encased in a tough protective coating. Spores are able to change back into vegetative bacteria, and so it is important to ensure that spores are ANTIBIOTIC RESISTANCE LAB|MICR 290 M08 PAGE 6 MODULE 08 COMPANION GUIDE MICR 290 destroyed during the sterilization process. As these spores are so robust, they are excellent indicators to test whether a sterilization method is working properly. Scanning electron micrograph of Mycobacterium tuberculosis cells. Mycobacteria, such as Mycobacterium tuberculosis (the causative agent of tuberculosis), are a group of bacteria that are very difficult to kill, and are sometimes used as biological indicators. Through noticeable colour changes, biological indicators demonstrate whether sterilization attempts have succeeded or failed.1 When a biological indicator is used to test the efficacy of an autoclave, the indicator is added to the autoclave load along with the equipment or material that is being sterilized. After the autoclave is finished, the biological indicator is incubated under conditions that would allow it to grow. If the spores grow under these conditions (often observed by a colour change), this means that the sterilization process has failed. Chemical Indicators Chemical indicators are tests that undergo some physical or chemical change when treated to the sterilization conditions. For example, chemical indicators might change colour if kept at a high temperature for a long enough time. The chemical indicator is placed in the autoclave along with the equipment that is being sterilized. If the chemical indicator does not respond as expected (e.g. change colour), this suggests that the autoclave did not achieve the expected temperature and pressure. As a result, this suggests that the autoclave cycle failed, and that the material in the autoclave is not sterile. ANTIBIOTIC RESISTANCE LAB|MICR 290 M08 PAGE 7 MODULE 08 COMPANION GUIDE MICR 290 A chemical indicator strip.2 Levels of Disinfection Although there are many different methods for disinfection, the process of disinfection is commonly organized into three separate levels: High-level disinfection Intermediate-level disinfection Low-level disinfection These different levels are broadly classified based on their ability to destroy vegetative bacteria, mycobacteria, and bacterial spores, as well as viruses and fungi. High-Level Disinfection This process destroys or inactivates all vegetative bacteria, including mycobacteria, and also destroys fungi and viruses. While high-level disinfection destroys some bacterial spores, it cannot reliably destroy large numbers of spores (in contrast to sterilization methods). Intermediate-Level Disinfection This process destroys vegetative bacteria, including mycobacteria, and most viruses and fungi. However, this level of disinfection does not kill bacterial spores. Low-Level Disinfection This process destroys vegetative bacteria and some fungi and viruses. However, it does not kill mycobacteria or spores. In a healthcare setting, not all equipment and patient-care items need to be sterilized or disinfected to the same level of rigour. Surgical instruments must be carefully sterilized to avoid causing infections, while blood pressure cuffs may only need to be disinfected. The Spaulding Classification System The choice of an appropriate method, whether sterilization or a particular level of disinfection, depends on the use of a particular piece of equipment. To help healthcare facilities such as hospitals decide how to sterilize or disinfect specific pieces of medical equipment, Dr. Earle Spaulding devised a ANTIBIOTIC RESISTANCE LAB|MICR 290 M08 PAGE 8 MODULE 08 COMPANION GUIDE MICR 290 classification system to streamline this process. The Spaulding classification system distinguishes medical items as: Critical items Semicritical items Noncritical items Learn more about each class of the Spaulding system. CRITICAL ITEMS Critical items are those that pose a high risk of infection if they are contaminated by vegetative bacteria, bacterial spores, or other microorganisms. These items come in contact with sterile tissue and/or the vascular system, and their contamination could lead to the transmission of disease. SEMICRITICAL ITEMS Semicritical items come in contact with mucous membranes or non-intact skin. As such, these items should be free of microorganisms, although it is permissible for there to be a small number of bacterial spores. NONCRITICAL ITEMS Noncritical items come in contact with intact skin, but not with mucous membranes. As intact skin represents an effective barrier to bacteria, it is not necessary that noncritical items be sterile. Based on what you have learned so far, match the appropriate level of control with the corresponding Spaulding classification item. Semicritical Items These Items must be sterilized before use, killing all microorganisms and spores Critical Items At minimum, these should be treated with high-level disinfectants, eliminating most spores Noncritical Items Typical, low-level disinfectants are sufficient for decontaminating these items Feedback: Critical Items These Items must be sterilized before use, killing all microorganisms and spores Semicritical Items At minimum, these should be treated with high-level disinfectants, eliminating most spores Noncritical Items Typical, low-level disinfectants are sufficient for decontaminating these items In this section, you learned about four processes that are commonly used to control bacterial growth (sterilization, disinfection, antisepsis, and sanitization), and the general mechanisms by which these agents function. You also learned about different types of indicators and how they are used to test the efficacy of a sterilization method. You were then introduced to the different levels of disinfection, and a classification system that can be used to determine which bacterial control method is the most appropriate for decontaminating a piece of medical equipment. References ANTIBIOTIC RESISTANCE LAB|MICR 290 M08 PAGE 9 MODULE 08 COMPANION GUIDE MICR 290 1. Weber Scientific. (n.d.). EZTest Biological Indicators (Mesa Labs). Retrieved October 20, 2020, from https://www.weberscientific.com/eztest-biological-indicators-mesa-labs 2. Steris Healthcare. (n.d.). Celerity HP Chemical Indicator. Retrieved October 20, 2020, from https://www.steris.com/healthcare/products/sterility-assurance-and-monitoring/chemical- indicators/celerity-hp-chemical-indicator 3. Medgadget. (2019). Surgical Instruments Market Outlook (2019 – 2025) – Industry Growth Factors, Market Revenue and More: QY Research. Retrieved October 20, 2020, from https://www.medgadget.com/2019/02/surgical-instruments-market-outlook-2019-2025- industry-growth-factors-market-revenue-and-more-qy-research.html 4. 1800Wheelchair. (n.d.). Respiratory Care Therapy Equipment & Supplies. Retrieved October 20, 2020, from https://www.1800wheelchair.ca/category/304/respiratory-care/ 5. Medgadget. (2020). Blood Pressure Cuffs Market Significant Applications, Largest Share, 2020, Data Processing & Analysis, Size Estimation, Growth Factors and Global Industry Trends to Forecast by 2027. Retrieved October 20, 2020, from https://www.medgadget.com/2020/03/blood-pressure-cuffs-market-significant-applications- largest-share-2020-data-processing-analysis-size-estimation-growth-factors-and-global- industry-trends-to-forecast-by-2027.html End of Section 01 ANTIBIOTIC RESISTANCE LAB|MICR 290 M08 PAGE 10 MODULE 08 COMPANION GUIDE MICR 290 SECTION 02: CHEMICAL METHODS OF CONTROL There are many different chemicals that can be used to kill bacterial cells on surfaces, instruments, and living tissues. In this section, you will learn about several chemical agents that are commonly used in the lab, healthcare facilities, or in household products. Most of these chemicals are used as disinfectants or antiseptics, and not as sterilants. Those chemicals that are disinfectants or antiseptics tend to be safer to use than sterilants, with fewer toxic effects on living tissue. In this section, you will learn about five common types of chemicals used to control bacterial growth, along with some other frequently used methods. Alcohol Solutions Bleach Phenols and Bisphenols Aldehydes Ethylene Oxide Other Chemical Methods Alcohol Solutions Alcohols are convenient chemical disinfectants because they evaporate after use and do not need to be dried. Many lab instruments and bench surfaces are regularly disinfected using 70% ethanol. Ethanol is an alcohol with both hydrophobic and hydrophilic characteristics. Chemical structure of ethanol, for your interest. Some major brands of hand sanitizer, such as Purell, contain 70% ethanol. Chemical structure of isopropanol, for your interest.1 Solutions of isopropanol, another alcohol, can also be used as disinfectants and antiseptics. Similar to ethanol, isopropanol has hydrophilic and hydrophobic properties. Using what you have learned about chemical control agents so far, answer the question. ANTIBIOTIC RESISTANCE LAB|MICR 290 M08 PAGE 11 MODULE 08 COMPANION GUIDE MICR 290 How might the chemical properties of ethanol and isopropanol relate to their function in controlling the growth of bacteria? Why might they be more or less effective than water alone? Dr. Lohans’ Response: While water alone is very polar, ethanol and isopropanol are somewhat hydrophobic. If a protein is in an alcoholic solution, the hydrophobic amino acids that are buried in the protein structure may become exposed. This can result in the unfolding of the protein, causing it to lose its activity. Bacteria have many important proteins on their surfaces, which can be denatured by alcohols. In addition, because of their relative hydrophobicity, alcohols can disrupt bacterial lipid membranes, helping to dissolve the lipids. Note that ethanol is only effective when used at a concentration above 70%. Interestingly, while many brands of mouthwash contain ethanol, the concentration of ethanol is too low to kill bacteria (< 30%). Instead, the ethanol is used to help dissolve hydrophobic ingredients in the mouthwash, and it is these ingredients which have antiseptic properties. Bleach Bleach is a common alternative to ethanol for disinfecting surfaces and instruments in the lab. Outside the lab, bleach is used as a disinfectant at home, in hospitals, and in medical clinics. Hypochlorous acid, a component of bleach, is very reactive, and is able to chemically modify proteins, D N A, and lipids. Hypochlorous acid can react with the side chains of many different amino acids found in proteins and can modify the structure of the nitrogenous bases found in D N A molecules. As the chemical structures of amino acids and nucleotides are central to the functions of proteins and D N A, their modification by hypochlorous acid is extremely disruptive to bacterial cells. These chemical changes lead to extensive protein unfolding and aggregation, preventing proteins from functioning properly. Diagram of the effects of hypochlorous acid on a bacterial cell. 2 Phenols and Bisphenols Chemical structure of phenol, for your interest. ANTIBIOTIC RESISTANCE LAB|MICR 290 M08 PAGE 12 MODULE 08 COMPANION GUIDE MICR 290 Phenols and bisphenols are molecules that contain a phenol functional group, which consists of a benzene ring with an O H group attached. In the 19th century, British surgeon John Lister observed that disinfecting surgical instruments with phenol decreased the risk of bacterial infection, helping to set the stage for infection control in the operating room. Phenols and bisphenols are very hydrophobic molecules that can disrupt the lipid membranes of bacteria. This results in the leakage of important components from within the cell, and causes the lysis of the cell membrane. The antiseptic mouthwash Listerine was named after John Lister. However, Listerine does not contain phenol. Phenol is corrosive and irritating to skin, and it has waned in popularity as an antiseptic and disinfectant. For this reason, derivatives of phenol are more commonly used, and are key components in many household disinfectants. Switch between o-phenylphenol and triclosan, two phenol derivatives used as disinfectants. o-Phenylphenol, a phenol derivative, is the main ingredient in many formulations of Lysol, a common cleaning and disinfectant product. Triclosan is a bisphenol that is used in toothpastes to help prevent the formation of plaque. Triclosan was extremely widely applied as a disinfectant in many household products, such as soaps, toothpastes, cutting boards, and knife handles, before its use was restricted to a small number of applications (e.g., toothpastes). Hypothesize why the use of triclosan has become limited. Then, listen to Dr. Lohans explain this evolution in the use of this disinfectant. (1:00) Start of Audio Transcript: As you’ve just read, triclosan has been used in a diverse range of household products, products used for personal hygiene, and also as a disinfectant in hospitals. However, this widespread use of triclosan has resulted in the emergence of bacteria that are resistant to its antibacterial activity. These triclosan-resistant bacteria tend to use efflux pumps, which expel triclosan from within the bacterial cell. As a result of this, the United States banned the use of triclosan in many consumer products. While the Canadian government did not observe the same impact of triclosan on the emergence of resistant bacteria, the use of this agent was also found to be harmful to the environment. Additionally, it was also seen that triclosan increased the ability of some pathogenic bacterial species to colonize human tissues. Based on all of these considerations, triclosan has largely fallen out of favour, although it is still currently used. ANTIBIOTIC RESISTANCE LAB|MICR 290 M08 PAGE 13 MODULE 08 COMPANION GUIDE MICR 290 End of Audio Transcript. Aldehydes Aldehydes, such as formaldehyde and glutaraldehyde, are very effective disinfectants. These molecules contain an aldehyde functional group, in which a carbon has a double bond to an oxygen, a single bond to a hydrogen, and a single bond to another carbon or hydrogen. Learn more about some of the aldehydes used as disinfectants. FORMALDEHYDE Formaldehyde has often been used to preserve biological specimens. This molecule reacts with the side chains of different amino acids in proteins, forming cross-links between them. This inactivates the proteins and enzymes, preventing them from degrading the different components of the specimen. For the same reason, formaldehyde is also very effective at killing bacteria. Chemical structure of formaldehyde, for your interest. GLUTARALDEHYDE Glutaraldehyde reacts with proteins in a similar way to formaldehyde, and is more effective as a disinfectant. It can be used to disinfect medical instruments, killing all of the bacteria and viruses. Longer soaking can even kill spores, and as such, glutaraldehyde can be considered to be a sterilant. However, the length of time required to kill spores with glutaraldehyde is often impractical. In addition, there are concerns that glutaraldehyde has possible mutagenic and carcinogenic effects. Chemical structure of glutaraldehyde, for your interest. ORTHO-PHTHALALDEHYDE Another aldehyde, ortho-phthalaldehyde, has been shown to be a very effective disinfectant, and is widely used to disinfect semi-critical items. Chemical structure of ortho-phthalaldehyde, for your interest. ANTIBIOTIC RESISTANCE LAB|MICR 290 M08 PAGE 14 MODULE 08 COMPANION GUIDE MICR 290 Ethylene Oxide As many medical applications require that all spores be killed, sporicidal agents have great importance in healthcare settings. Ethylene oxide is a small molecule consisting of a three-membered ring with two carbons and one oxygen. Similar to what you learned regarding the beta-lactam antibiotics (which contain a four-membered ring) in Module 02, the three-membered ring in ethylene oxide is very strained and very reactive. As a result, ethylene oxide can react with nucleotides and the side chains of certain amino acids, forming covalent adducts. This is known as an alkylation reaction, and the alkylation of D N A and proteins dramatically interferes with their cellular functions, leading to bacterial death. Chemical structure of ethylene oxide, for your interest. The alkylation of DNA by ethylene oxide.3 Photograph of an ethylene oxide sterilizer.4 Ethylene oxide is considered to be a sterilant, as it can kill bacterial spores and bacterial cells. It reacts with all of the proteins it encounters, including those in living tissues. As ethylene oxide is a gas, this makes it challenging to work with safely. Therefore, healthcare facilities will often use an ethylene oxide sterilizer, which is a sealed chamber that can be filled with ethylene oxide. Other Chemical Methods of Control ANTIBIOTIC RESISTANCE LAB|MICR 290 M08 PAGE 15 MODULE 08 COMPANION GUIDE MICR 290 Learn about other chemical methods of control. SOAPS AND DETERGENTS One of the most routine ways of controlling bacterial growth on skin is through the use of soaps and detergents. However, soap isn’t considered to be an antiseptic, and does not kill most microbes. Instead, soap can help mechanically remove bacteria and other microorganisms from skin, disrupting the oily film on the skin surface and washing off the bacteria. Illustration of the process by which soap molecules (orange) remove dirt and debris during handwashing.5 ANTIBIOTICS As already discussed, while antibiotics are chemicals used to control bacterial growth, they are usually considered separately from sterilants, disinfectants, antiseptics, and sanitizers. Antibiotics usually target specific bacterial pathways, while the other chemical methods discussed in this section have more general mechanisms of action. Antibiotics are used to control bacterial growth. Use what you’ve learned in this section about the chemical methods of control to answer the question. What are the most common mechanisms by which chemical agents are able to control bacterial growth? Use examples of specific chemicals in your answer. Feedback: Dr. Lohans’ Response: Chemical methods of control tend to work through a few general mechanisms. They can: ANTIBIOTIC RESISTANCE LAB|MICR 290 M08 PAGE 16 MODULE 08 COMPANION GUIDE MICR 290 Denature and/or damage proteins through hydrophobic interactions, or through reaction with the side chains of amino acids (e.g. alcohol, bleach, aldehydes, and ethylene oxide) Disrupt lipid membranes (e.g. alcohols and phenols) React with D N A to modify the structures of the nitrogenous bases (e.g. bleach and ethylene oxide) As you have learned in this section, there are a wide variety of chemical methods for controlling bacterial growth, and there are many more varieties than were introduced here. These chemical methods have different levels of efficacy, and some are used as disinfectants or antiseptics, while others are used as sterilants. The efficacy of these chemicals at killing bacteria can be determined using methods such as the disk diffusion test, which is described in Section 04 of this module. References: 1. U.S. National Library of Medicine. (n.d.) Isopropyl alcohol. Retrieved October 20, 2020, from https://chem.nlm.nih.gov/chemidplus/rn/67-63-0 2. da Cruz Nizer, W. S., Inkovskiy, V., & Overhage, J. (2020). Surviving Reactive Chlorine Stress: Responses of Gram-Negative Bacteria to Hypochlorous Acid. Microorganisms, 8(8), 1220. Retrieved October 20, 2020, from https://doi.org/10.3390/microorganisms8081220 3. TECHEFY. (2017, April 15). Ethylene Oxide as a Sterilizing Agent for Sensitive Medical Equipment. Retrieved October 20, 2020, from http://www.techefy.com/ethylene-oxide-as-a- sterilizing-agent-for-sensitive-medical-equipment/ 4. Lexamed. (n.d.). EO Sterilization. Retrieved October 20, 2020, from https://www.businesswire.com/news/home/20150604005062/en/Ethylene-Oxide-Sterilizer- with-Unique-Cycle-Customization-Software-Helps-Give-Life-Science-Companies-More-Control 5. Gerhardt, M. (2017, January 10). Say Goodbye to Antibacterial Soaps: Why the FDA is banning a household item. Science in the News. Retrieved October 20, 2020, from http://sitn.hms.harvard.edu/flash/2017/say-goodbye-antibacterial-soaps-fda-banning- household-item/ End of Section 02 ANTIBIOTIC RESISTANCE LAB|MICR 290 M08 PAGE 17 MODULE 08 COMPANION GUIDE MICR 290 SECTION 03: PHYSICAL METHODS OF CONTROL As with the chemical methods, there are many physical methods that can be used to control bacterial growth, and you have already learned about several of them in previous modules. While chemical methods work by molecular means to target the molecules that make up the bacterial cell, physical methods use agents such as electromagnetic radiation, osmosis, and temperature to slow the growth of bacteria or to kill them outright. Temperature One of the simplest physical methods for controlling bacterial growth is simply by decreasing or increasing the temperature. Review a general overview on how temperature can be used to control bacterial growth. Low Temperatures At low temperatures, the metabolism of most bacteria slows so that they are unable to reproduce. For example, the temperature in most refrigerators (around 4 - 8 °C) inhibits the growth of most of the bacteria found in food. These low temperatures have a bacteriostatic effect, but do not kill most bacteria. However, some bacterial species are still able to grow slowly at low temperatures, and can even survive being frozen. The temperature in refrigerators stunts the growth of bacteria in food. 1 High Temperatures High temperatures can be used to kill bacteria, and there are several physical methods that use heat for controlling bacterial growth. For example, boiling water can be used to kill most vegetative bacteria. At this high temperature (100°C), the proteins in bacterial cells denature and aggregate, killing the bacterial cells. However, bacterial spores can be stable in boiling water, sometimes surviving these conditions for more than 20 hours. Boiling water can kill bacteria in vegetables. You will now explore more physical methods that use heat to control bacterial growth. Autoclaves You were previously introduced to the autoclave, a piece of equipment that uses high pressure steam to sterilize equipment and reagents. The use of high pressure allows autoclaves to operate at temperatures above 100 °C, and a temperature of 121 °C is often used for autoclaving. At this temperature, bacteria and bacterial spores are killed, ensuring that the contents of the autoclave are sterilized. Autoclaves are commonly used in hospitals, dental offices, and research labs. ANTIBIOTIC RESISTANCE LAB|MICR 290 M08 PAGE 18 MODULE 08 COMPANION GUIDE MICR 290 Photograph of an autoclave. Pasteurization Many food products cannot tolerate the high temperatures required for sterilization. In the 19th century, French microbiologist Louis Pasteur discovered that mild heating was sufficient to kill many of the bacteria responsible for food spoilage. This process, now known as pasteurization, is used to eliminate pathogenic bacteria from dairy products such as milk. While pasteurization decreases the total number of bacteria in milk, it does not kill many heat-resistant bacteria; however, these bacteria generally do not cause disease or cause food to spoil. High-temperature short-time (H T S T) pasteurization is commonly used to treat milk, treating it at approximately 72 °C for 15 seconds. While most milk available in the store has been pasteurized, it is also possible to purchase ultra-high temperature (U H T) processed milk, which is sterile. U H T milk can be stored for many months at room temperature, while pasteurized milk must be refrigerated and generally lasts only 1-2 weeks. Dry Heat Boiling, autoclaving, pasteurization, and U H T processing all use moist heat to kill bacteria. Dry heat can also be used to sterilize surfaces and equipment. Most commonly, this involves the use of a flame to kill bacteria. For example, a Bunsen burner is often used in the microbiology lab to sterilize pieces of lab equipment (e.g. inoculation loops) and the openings of sterile bottles while working aseptically. Dry heat without a flame can also kill bacteria, as in hot-air sterilization. Items can be sterilized by placing them in an oven and treating them at high temperatures for a long period of time. Filtration While many lab solutions and reagents can be sterilized by autoclaving, not all of them are stable at high temperatures, which may cause them to react or degrade. As an alternative, solutions can be filter-sterilized, passing them through a filter with very small pores. These pores are often 0.22 μm or 0.45 μm in size, and are small enough that bacteria cannot pass through. If a solution is filtered into a sterile container, the filtrate will be sterile. ANTIBIOTIC RESISTANCE LAB|MICR 290 M08 PAGE 19 MODULE 08 COMPANION GUIDE MICR 290 Air can also be filtered to remove microbes, which may be important in environments such as the operating room in a hospital. High-efficiency particulate air (HEPA) filters can remove almost all airborne microorganisms that are larger than 0.3 μm. HEPA filters are also installed in some biosafety cabinets used to handle biohazards in research labs, preventing them from spreading in the environment, and protecting the researchers that work with them. Electromagnetic Radiation The electromagnetic spectrum with the spectrum of visible light expanded in the bottom panel. 7 Electromagnetic radiation can be used to kill microorganisms such as bacteria. There are two kinds of sterilizing radiation: ionizing radiation and nonionizing radiation. These different kinds of electromagnetic radiation differ in terms of their wavelengths and relative energies. Compare the two types of sterilizing radiation. IONIZING RADIATION Ionizing radiation is high energy electromagnetic radiation, which includes gamma rays and X-rays. This form of radiation can ionize water, resulting in the formation of extremely reactive hydroxyl radicals. These radicals react with many components in the cell, and in particular they can damage D N A. This can lead to mutations; a sufficient number of mutations will lead to the death of bacterial cells. Gamma rays can penetrate deeply into samples, but require a long exposure time to fully sterilize a sample. Ionizing radiation is used to sterilize lab equipment and disposable medical supplies. NONIONIZING RADIATION ANTIBIOTIC RESISTANCE LAB|MICR 290 M08 PAGE 20 MODULE 08 COMPANION GUIDE MICR 290 Nonionizing radiation is lower in energy than ionizing radiation, and does not lead to the formation of hydroxyl radicals. Ultraviolet (U V) light, a kind of nonionizing radiation, can damage the D N A in bacterial cells, causing the formation of chemical bonds between adjacent nucleotides. This prevents the replication of D N A, preventing bacteria from dividing. U V light does not penetrate as deeply as gamma rays, and can only kill bacteria that are exposed on the surface of a piece of equipment. U V lamps are sometimes used in hospital rooms, operating rooms, and in biosafety cabinets to help sterilize these environments when they are not in use. Osmotic Pressure In Module 02, you learned that the bacterial cell wall helps bacteria resist changes in osmotic pressure (i.e. the pressure that results when the concentration of salts and small molecules inside the bacterial cell is different from their concentration outside the bacterial cell). It is possible to overwhelm a bacterial cell by placing it in an environment with an extremely high osmotic pressure (i.e. a hypertonic solution). In an environment with high amounts of salt or small molecules, the water inside a bacterial cell will be drawn out, and the bacteria will be unable to grow. Comparison between a bacterial cell in an isotonic solution and a bacterial cell in a hypertonic solution.8 Bacterial cells require moisture to grow and survive. Related to those methods that are based on osmotic pressure, bacterial growth can also be inhibited through desiccation, which refers to processes that remove moisture from the bacterial cell. For example, when food products such as coffee are freeze dried, the moisture is removed, which prevents bacterial growth. Desiccation does not necessarily kill bacteria as addition of water to the desiccated bacteria allows them to resume growing and replicating. Meats are cured by treating them with very high salt concentrations. This salt draws the water out of the meat, and also draws the water out of any bacterial cells that are present. This prevents the ANTIBIOTIC RESISTANCE LAB|MICR 290 M08 PAGE 21 MODULE 08 COMPANION GUIDE MICR 290 bacteria from growing and spoiling the meat. Similar principles explain why high sugar concentrations are used to preserve jams, and why honey can be stored for a long period of time. Using what you have learned about the different types of physical methods for controlling bacterial growth, match each term with the appropriate description. Choices: Bunsen Burner Desiccation HEPA Filters Pasteurization Curing Autoclaves Equipment used to eliminate airborne microorganisms A piece of equipment that utilizes dry heat for sterilization Removing moisture to prevent bacterial growth A process used to eliminate pathogenic bacteria from food products using high temperatures Sterilizes equipment using high pressure steam A method of preventing bacterial growth using high salt concentrations Feedback: A process used to eliminate pathogenic bacteria from food products using high Pasteurization temperatures A piece of equipment that utilizes dry heat for sterilization Bunsen Burner Equipment used to eliminate airborne microorganisms HEPA Filters A method of preventing bacterial growth using high salt concentrations Curing Removing moisture to prevent bacterial growth Desiccation Sterilizes equipment using high pressure steam Autoclaves As you’ve learned in this section, there are many different physical methods that can be used to control bacterial growth. Selection of the best method depends on the type of material or equipment that is being sterilized or preserved. Some methods are suitable for killing bacteria in liquid samples, while others are better for killing bacteria present on solid surfaces. References: 1. Authorized Service. (2020, June 22). Is My Fridge Overheating? Why Your Refrigerator is Hot Underneath. Retrieved October 20, 2020, from https://authorizedco.com/refrigerator- repair/is-my-fridge-overheating-why-your-refrigerator-is-hot-underneath/ 2. Hansen’s Dairy. (2013, April 2). Raw milk vs. Pasteurized milk… Retrieved October 20, 2020, from https://hansendairy.wordpress.com/2013/04/02/raw-milk-vs-pasteurized-milk/ 3. Gay Lea Foods Co-Operative Limited. (n.d.). Fluids - UHT Milk & Cream. Retrieved October 20, 2020, from https://www.gaylea.com/foodservice/products/fluids-uht-milk-cream/ ANTIBIOTIC RESISTANCE LAB|MICR 290 M08 PAGE 22 MODULE 08 COMPANION GUIDE MICR 290 4. Bryant, S. (2018, July 17). What equipment do I need to perform a vacuum filtration? The Laboratory People. Retrieved October 20, 2020, from https://camblab.info/what-equipment- do-i-need-to-perform-a-vacuum-filtration/ 5. ISC Sales. (n.d.). 12x12x11.5 High-Efficiency HEPA Air Filter - 99.97%, Koch H61A1X1. Retrieved October 20, 2020, from https://iscsales.com/item/12x12x11-1-2-biomax-hepa-filter-99-97-high- efficiency-k1121212-sbm/ 6. Alibaba.Com. (n.d.). Class II A2-1000D Cheap Price Biological Safety Cabinet Biosafety Cabinets Supplier, View Biosafety Cabinets Supplier. Retrieved October 20, 2020, from https://kenton.en.alibaba.com/product/62265727638- 802947827/Class_II_A2_1000D_Cheap_Price_Biological_Safety_Cabinet_Biosafety_Cabinets_Sup plier.html 7. Environmental Chemistry Portfolio. (n.d.). Electromagnetic Spectrum. Retrieved October 20, 2020, from https://sites.google.com/site/winzevscience2015/notes/electromagnetic-spectrum 8. Liberman, B. (2018). Three methods of forward osmosis cleaning for RO membranes. Desalination, 431, 22–26. Retrieved October 20, 2020, from https://doi.org/10.1016/j.desal.2017.11.023 End of Section 03 ANTIBIOTIC RESISTANCE LAB|MICR 290 M08 PAGE 23 MODULE 08 COMPANION GUIDE MICR 290 SECTION 04: TESTING ANTIBIOTIC SUSCEPTIBILITY So far in this module, you have learned about chemical and physical methods that are used to control bacterial growth. You have also learned that antibiotics can be used to selectively inhibit or kill bacteria. Chemical agents and antibiotics have varying levels of activity against different kinds of bacteria, and in order to decide on the best chemical agent or antibiotic to use, the agent/antibiotic must first be tested in the lab. This section will introduce you to the disk diffusion test, the epsilometer test, and the broth microdilution test, all of which can be employed to test the efficacy of antibiotics and other chemical agents. Disk Diffusion Test One of the most popular methods for measuring antibiotic activity is the disk diffusion test, also known as the Kirby-Bauer test. This test uses small pieces of filter paper that are impregnated with an antibiotic. An agar plate is coated with a very thin layer of the indicator strain of bacteria (i.e. the bacteria that is being tested for antibiotic susceptibility), the antibiotic disks are placed on top, and the plate is incubated. If the indicator bacteria is susceptible to the antibiotic, there will be a clear zone of inhibition around the disk. If the bacteria is fully resistant to the antibiotic, the area around the disk will be cloudy with bacterial growth. The zone diameter, generally measured in millimetres, can be related to the susceptibility of the indicator bacteria to the antibiotic. Switch between a diagram and a photograph of a disk diffusion test. A disk diffusion test using three different types of antibiotic disks labelled A, B, and C. Each antibiotic inhibits bacterial growth to a different extent. ANTIBIOTIC RESISTANCE LAB|MICR 290 M08 PAGE 24 MODULE 08 COMPANION GUIDE MICR 290 The results of a disk diffusion test, measuring the susceptibility of the indicator bacteria to nine different antibiotics.1 Use the image provided and your current knowledge of the disk diffusion test to answer the question. Staphylococcus aureus tested with two different antibiotics.2 Which antibiotic is the indicator bacteria least susceptible to? a) A b) B c) The indicator bacteria is equally susceptible to both antibiotics d) The indicator bacteria is not susceptible to either antibiotic Feedback: B. Dr. Lohans’ Response: There is a larger zone of inhibition around the disk with antibiotic A than there is around the disk with antibiotic B, which suggests the indicator bacteria is less susceptible to antibiotic B. As you can appreciate from the provided images, a major advantage of the disk diffusion test is that multiple antibiotics can be tested on the same agar plate. Disks containing different antibiotics ANTIBIOTIC RESISTANCE LAB|MICR 290 M08 PAGE 25 MODULE 08 COMPANION GUIDE MICR 290 are placed on different parts of the agar surface. The diameter of each zone of clearing can be measured, allowing the antibiotic susceptibility of the indicator strain to a broad panel of antibiotics to be quickly determined. The protocol for a disk diffusion test is simple, fast, and cheap. Learn the experimental steps for setting up a disk diffusion test. The experimental steps of a disk diffusion test.3 1. Indicator Bacteria Preparation First, a culture of the indicator bacteria is grown in liquid media. 2. Agar Plate Preparation Once the culture is grown, a sterile swab is used to spread the culture over the surface of an agar plate. It is important to swab the whole surface of the agar plate, so that the indicator bacteria will grow everywhere on the agar (i.e. so that the plate will become confluent). 3. Disk Placement After the agar plate has been swabbed and allowed to dry, antibiotic-impregnated disks are applied to the surface of the agar plate (employing aseptic technique!). Then, the plate is incubated under suitable conditions to allow the indicator bacteria to grow. 4. Zone of Clearing Measurement Once the indicator bacteria has grown, any zones of clearing that appear can be measured. As you will learn in Section 05 of this module, there are strict guidelines for properly carrying out disk diffusion assays. By following these guidelines, the zone diameters from these tests can be compared to published numbers to determine their clinical significance in terms of antibiotic resistance and susceptibility. Minimum Inhibitory Concentration Although disk diffusion tests give a quantitative measure of the antibiotic susceptibility of an indicator bacteria (i.e. a zone diameter), this value by itself is not very meaningful. It is only useful when compared to other results from disk diffusion tests. A more generally useful way of measuring the antibiotic susceptibility of a bacterial strain is by determining a value known as a minimum inhibitory concentration (M I C). The M I C is the smallest concentration of antibiotic that fully inhibits the growth of a particular bacterial strain. An M I C is particular to the combination of a specific antibiotic and a specific strain of bacteria. ANTIBIOTIC RESISTANCE LAB|MICR 290 M08 PAGE 26 MODULE 08 COMPANION GUIDE MICR 290 Use what you have learned in this section to answer the question. Which of the statements are true? Select all that apply. a) The M I C is inversely related to the susceptibility of this strain to the antibiotic. b) The higher the M I C value for an antibiotic, the less antibiotic is needed to fully inhibit the bacteria. c) If the bacterial strain becomes resistant to the antibiotic, the MIC of this antibiotic for the resistant strain will be greater. Feedback: A and C. Dr. Lohans’ Response: The lower the M I C value for an antibiotic, the less antibiotic is needed to fully inhibit the bacteria, and the more susceptible the bacteria is to the antibiotic. If a bacterial strain becomes resistant to the antibiotic, a higher concentration of antibiotic will be needed to fully inhibit bacterial growth, and the M I C will become greater. Epsilometer Test Several antibiotic susceptibility tests can be used to determine M I C values. One of the easiest methods is the Epsilometer test (E test), which is conceptually very similar to the disk diffusion tests. In an E test, an antibiotic-impregnated strip is used to inhibit the growth of an indicator strain on an agar plate. While Kirby-Bauer disks contain a uniform amount of antibiotic throughout the disk, E test strips contain a gradient of antibiotic. One end of the E test strip contains a large amount of antibiotic, and the other end contains a small amount of antibiotic. As in a disk diffusion test, the surface of an agar plate is first swabbed with a very thin layer of the culture of the indicator bacterial strain. Then, an E test strip is applied to the surface of the agar plate, and the plate is incubated to allow the indicator strain to grow. The results of an E test. The numbers on the E strip indicate antibiotic concentration in µg/m L.4 If the indicator strain is susceptible to the antibiotic, there will be a zone of clearing around the E test strip. However, this zone will not be uniform around the entire strip. The zone will be largest around ANTIBIOTIC RESISTANCE LAB|MICR 290 M08 PAGE 27 MODULE 08 COMPANION GUIDE MICR 290 the end of the strip that contains the greatest amount of antibiotic, and will decrease in size along the strip as the local antibiotic concentration decreases. The end of the E test strip with the lowest amount of antibiotic typically does not kill the indicator bacteria, and there is no zone of clearing at this end. As a result, the zone of clearing will intersect the strip somewhere in the middle. This offers a very easy way to determine the MIC for the antibiotic against the indicator bacteria. Use the images provided and your current knowledge of the E test to answer the questions. Testing the susceptibility of Streptococcus pneumoniae to the β-lactam antibiotic cefotaxime.5 1 of 2: What is the MIC for cefotaxime against S. pneumoniae? a) 0.15 µg/m L b) 0.12 µg/m L c) 0.5 µg/m L d) 2.0 µg/m L Feedback: 0.5 µg/m L Dr. Lohans’ Response: Recall that the M I C is the lowest antibiotic concentration that completely inhibits bacterial growth; this corresponds to the lowest antibiotic concentration on the E test strip that gives a zone of clearing. The M I C can be determined by looking at the concentration marking on the E test strip where the zone of clearing appears. For this example, the M I C is 0.5 µg/m L. ANTIBIOTIC RESISTANCE LAB|MICR 290 M08 PAGE 28 MODULE 08 COMPANION GUIDE MICR 290 Enterococcus faecium tested with the antibiotic teicoplanin.6 Enterococcus faecium tested with the antibiotic vancomycin.7 2 of 2: Given the E test results shown, which statements are correct regarding the antibiotic susceptibility of E. faecium? Select all that apply. a) E. faecium is equally susceptible to vancomycin and teicoplanin. b) The M I C of vancomycin is 16 µg/m L. c) The M I C of teicoplanin is 16 µg/m L. d) E. faecium is less susceptible to teicoplanin. e) E. faecium is resistant to vancomycin at concentrations 67 d) n 7 Protein 1 has a p I < 7 Feedback: Protein 3 has a p I = 7 The protein will have a net neutral charge at p H = 7 Protein 2 has a p I > 7 The protein will have a net positive charge at p H = 7 Protein 1 has a p I < 7 The protein will have a net negative charge at p H = 7 In a mixture of proteins, some proteins will be positively charged, some will be negatively charged, and some will be neutral. These different net charges can be used as the basis for separating these proteins through ion exchange chromatography. Ion exchange chromatography uses an electrostatically charged stationary phase (i.e. ion exchange resin). There are four main types of ion exchange resin: Strong cation exchange resin has a strong negative charge. Weak cation exchange resin has a weak negative charge. Strong anion exchange resin has a strong positive charge. Weak anion exchange resin has a weak positive charge. Note that the names of these resins do not correspond to their charge. Although cations are positively charged, cation exchange resin is negatively charged. Instead, these names refer to the charges of the molecules that bind to the resin. Cations bind to cation exchange resin, and anions bind to anion exchange resin. ANTIBIOTIC RESISTANCE LAB|MICR 290 M05 PAGE 52 MODULE 05 COMPANION GUIDE MICR 290 Comparison of anion and cation exchange resins. The resin is represented by the filled circles. Ion exchange resin is used to purify proteins based on electrostatic interactions. Proteins with a net negative charge will bind to anion exchange resins, but not to cation exchange resins. Similarly, positively charged proteins will bind to cation exchange resins, but not to anion exchange resins. Diagram of a cation exchange column. The strength of the binding interaction between proteins and ion exchange resin depends on the ionic strength of the protein buffer. The ionic strength is related to the amount of salt in the buffer; the higher the salt concentration, the higher the ionic strength. The most common salt used in protein buffers is sodium chloride (N a C l), which dissociates to sodium cations and chloride anions in solution. Similar to proteins, ions in the buffer interact with ion exchange resin. These salt ions compete with proteins for binding to the charged groups on the resin. In a buffer with a low ionic strength, the concentration of salt ions will be low, and there will not be enough salt ions to compete effectively with the protein for binding to the resin. In a buffer with a high ionic strength, the concentration of salt ions will be high, and they can outcompete the protein for binding to the resin. ANTIBIOTIC RESISTANCE LAB|MICR 290 M05 PAGE 53 MODULE 05 COMPANION GUIDE MICR 290 Answer the question to consolidate your understanding of buffers and p H and their impact on ion exchange chromatography. Consider the purification of a positively charged recombinant protein by cation exchange chromatography. Which p H and ionic strength would you choose for your buffer? a) Buffer with high p H, low salt concentration b) Buffer with high p H, high salt concentration c) Buffer with low p H, low salt concentration d) Buffer with low p H, high salt concentration Feedback: c). To enhance the positive charge of this protein, a buffer with a low pH could be used for this experiment. In addition, the salt concentration in this buffer is kept low, resulting in a low ionic strength. Let’s call the buffer from this example Buffer A (low salt). A crude mixture of proteins including the desired recombinant protein is loaded onto a column of cation exchange resin. The column is washed with Buffer A and fractions are collected of the material that elutes. The positively charged recombinant protein will bind strongly to the resin under these conditions, as will any other positively charged protein impurities. However, the proteins that are neutral or negatively charged will travel quickly through the column and will be collected in the early fractions. In order to elute the recombinant protein from the column (and to separate it from the other positively charged proteins), the composition of the buffer running through the column must be adjusted. Using the same scenario, answer the question using what you’ve learned about the buffers used in ion exchange chromatography. How might you adjust the buffer to elute the recombinant protein? a) Increase p H b) Decrease p H c) Increase salt concentration d) Decrease salt concentration Feedback: c). This could be accomplished by altering the p H of the buffer, by increasing the ionic strength of the buffer, or both. However, proteins often do not tolerate dramatic p H changes, and so increasing the ionic strength is the more common approach. In this example, a buffer of the same p H but with a higher concentration of salt is used. Let’s call the new buffer, Buffer B (high salt). ANTIBIOTIC RESISTANCE LAB|MICR 290 M05 PAGE 54 MODULE 05 COMPANION GUIDE MICR 290 If the cation exchange column was directly washed with Buffer B, all of the bound proteins would immediately elute, and the desired protein would be contaminated with all of the other positively charged proteins. Instead, the cation exchange column can be washed with a buffer gradient, such that the ionic strength of the buffer going through the column gradually increases over time. This can be accomplished by washing the column with mixtures of Buffers A and B such that the level of Buffer B increases and the level of Buffer A decreases gradually over time. These gradients can be: Stepwise, consisting of defined “steps” (e.g. a wash with a mixture of 90% Buffer A and 10% Buffer B, then a wash with 75% Buffer A and 25% Buffer B, and so on). Linear, in which the percent composition of Buffer B is gradually increased over time (e.g. the amount of Buffer B is increased by 1% every minute for 100 minutes). These kinds of gradients typically require the use of an F P L C with separate pumps for Buffers A and B. In this example, proteins that are less positively charged will elute earlier in the gradient. The proteins that are more strongly positively charged require a buffer with a stronger ionic strength to be eluted, requiring more Buffer B. Diagram of ion exchange chromatography, as discussed in the example. Test your mastery of the protein purification through chromatography by answering the short answer question. ANTIBIOTIC RESISTANCE LAB|MICR 290 M05 PAGE 55 MODULE 05 COMPANION GUIDE MICR 290 Compare and contrast the types of chromatography discussed in this section. When might you use ion exchange chromatography? When might you use nickel affinity chromatography? Feedback: Dr. Lohans’ Feedback: The best technique to use for a purification depends on the protein that is being purified. Nickel affinity chromatography is an extremely effective technique for purifying a protein, but requires that the protein has a His-tag. Size exclusion chromatography and ion exchange chromatography are also effective, but require that the protein being purified is a different size or has a different overall charge, respectively, than the other proteins in the mixture. Often, these techniques are done consecutively to obtain extremely pure protein, e.g. a protein can be purified first with nickel affinity chromatography and then further purified by ion exchange chromatography. In this section, you learned how chromatography can be used to separate proteins based on their structural and physical properties. Several chromatographic techniques are often used together in order to remove all of the impurities from a recombinant protein. In Module 05, you learned about the basics of cloning, using restriction enzymes and D N A ligase to insert a strand of D N A into a vector. Then, you learned several mechanisms by which transcription is regulated in bacteria, and saw how these can be applied to produce recombinant proteins in E. coli. Finally, you saw how recombinant proteins can be extracted from bacteria and purified by chromatography. Drawing on what you learned in the last two Modules, you now have all of the skills necessary to design a cloning experiment, using P C R to amplify a sequence of D N A and using restriction enzymes and D N A ligase to clone it into a vector. References: 1. Cox, M., Doudna, J., & O'Donnell, M. (2015). Molecular Biology (2nd ed.). W.H. Freeman and Company. 2. Biolabs, N. E. (n.d.). PMALTM. Retrieved September 28, 2020, from https://international.neb.com/products/protein-expression-and-purification- technologies/e-coli/pmal/pmal End of Section 05 End of Module 05 ANTIBIOTIC RESISTANCE LAB|MICR 290 M05 PAGE 56 MICR 290 oiw ANTIBIOTIC RESISTANCE LAB MODULE 04 POLYMERASE CHAIN REACTION Please note: This course was designed to be interacted and engaged with using the online modules. This Module Companion Guide is a resource created to complement the online slides. If there is a discrepancy between this guide and the online module, please refer to the module. How can you help protect the integrity and quality of your Queen’s University course? Do not distribute this Module Companion Guide to any students who are not enrolled in MICR 290 as it is a direct violation of the Academic Integrity Policy of Queen’s University. Students found in violation can face sanctions. For more information, please visit https://www.queensu.ca/academic- calendar/health-sciences/bhsc/. MODULE 04 COMPANION GUIDE MICR 290 TABLE OF CONTENTS SECTION 00: Module Overview............................................................................................................................ 3 SECTION 01: Introduction to the Structure of DNA........................................................................................... 4 SECTION 02: Polymerase Chain Reaction........................................................................................................... 9 SECTION 03: Sanger Sequencing.......................................................................................................................24 SECTION 04: qPCR,RT-PCR,and RT-qPCR...............................................................................................................30 ANTIBIOTIC RESISTANCE LAB|MICR 290 M04 PAGE 2 MODULE 04 COMPANION GUIDE MICR 290 SECTION 00: MODULE OVERVIEW In the last module, you learned about the different forms of DNA that occur in bacterial cells, and saw different mechanisms by which DNA is transferred between bacteria (e.g. vertical gene transfer and horizontal gene transfer). Now, you will learn how DNA is replicated by the enzyme DNA polymerase, and how this is the basis of the polymerase chain reaction (PCR) and Sanger sequencing. Watch the video of your instructor, Dr. Lohans, introducing the concepts that are covered in Module 04 (1:10). After completing this module, you will be able to: 1. Describe the major steps involved in PCR and the roles of the different reagents used. 2. Hypothesize how changes to the temperatures and times of a PCR thermocycling programme will impact the experimental outcome. 3. Design oligonucleotide primers in order to amplify a particular sequence of DNA by PCR, or to determine a particular sequence of DNA by Sanger sequencing. 4. Explain how Sanger sequencing differs from PCR. 5. Compare and contrast PCR, qPCR,RT-PCR,and RT-qPCR, and rationalize which one is appropriate to achieve a particular experimental objective. Page Link: https://player.vimeo.com/video/454100725 ANTIBIOTIC RESISTANCE LAB|MICR 290 M04 PAGE 3 MODULE 04 COMPANION GUIDE MICR 290 SECTION 01: INTRODUCTION TO THE STRUCTURE OF D NA To get a better understanding of how DNA replication works, you will review the chemical structure of DNA. DNA is a polymer made up of nucleotides. Each nucleotide consists of a deoxyribose sugar which is connected to a phosphate group and a nitrogenous base. There are four main nitrogenous bases in D NA: adenine (A), cytosine (C), guanine (G), and thymine (T). The combination of a deoxyribose sugar with a nitrogenous base (and without a phosphate group) is known as a nucleoside. Adjacent nucleotides are connected through a phosphodiester linkage, in which the deoxyribose sugar of one nucleotide is covalently attached to the phosphate group of another nucleotide. The general chemical structure of a nucleotide. Note that the chemical structure of the base differs between A, C, G, and T.1 The chemical structure of a nucleoside. Note that the chemical structure of the base differs between A, C, G, and T.1 Adjacent nucleotides are connected through a phosphodiester linkage, in which the deoxyribose sugar of one nucleotide is covalently attached to the phosphate group of another nucleotide. ANTIBIOTIC RESISTANCE LAB|MICR 290 M04 PAGE 4 MODULE 04 COMPANION GUIDE MICR 290 Adjacent nucleotides are linked through phosphodiester bonds.2 In cells, DNA is almost always double-stranded. The two strands of DNA are held together in part by hydrogen bond interactions between the nitrogenous bases. These base pair interactions occur between adenine and thymine (A and T), and between cytosine and guanine (C and G). The chemical structure of double-stranded DNA, highlighting the sugar-phosphate backbone and the hydrogen bonds between complementary nitrogenous bases.3 Take a moment to think about this question before continuing. It may be helpful to review the diagrams provided thus far in this section. Given the hydrogen bond interactions between the nitrogenous bases, can you think of a reason why the interaction between C and G is stronger than the interaction between A and T? Feedback: ANTIBIOTIC RESISTANCE LAB|MICR 290 M04 PAGE 5 MODULE 04 COMPANION GUIDE MICR 290 Dr. Lohans’ Feedback: There are two hydrogen bonds between A and T, and three hydrogen bonds between C and G. Therefore, the interaction between C and G is stronger than the interaction between A and T. The directionality of a strand of DNA is related to the chemical structure of the alternating deoxyribose sugars and phosphate groups that make up the DNA backbone. A strand of DNA has two ends: the 5ʹ end and the 3ʹ end (pronounced “five prime” and “three prime”). The names for the ends are based on the numbering of the carbons in the deoxyribose sugar. The phosphodiester linkage between adjacent nucleotides consists of a phosphate group covalently attached to carbon 5 of one deoxyribose sugar, and covalently attached to carbon 3 of the adjacent deoxyribose sugar. Carbon 5 of all of the deoxyribose sugars in a DNA strand is oriented towards one end (the 5ʹ end), and carbon 3 of all of these sugars is oriented towards the other end (the 3ʹ end). Double-stranded DNA is antiparallel, meaning that the two DNA strands are in opposite orientations: if one strand is oriented from 5ʹ to 3ʹ, the complementary strand will be oriented in the opposite direction (from 3ʹ to 5ʹ). Double-stranded molecule of DNA, showing the directionality of the DNA backbone.4 This directionality is extremely important, as it determines how two DNA strands bind together to form a double-stranded helix, how DNA is replicated by DNA polymerase, how DNA is transcribed by R N A polymerase, and how DNA interacts with proteins. DNA sequences are always written from the 5ʹ end to the 3ʹ end, unless indicated otherwise. ANTIBIOTIC RESISTANCE LAB|MICR 290 M04 PAGE 6 MODULE 04 COMPANION GUIDE MICR 290 Double-stranded DNA forms an antiparallel helix. Using what you have learned in this section about the chemical structure of DNA, select whether the provided statements are true or false. A nucleotide consists of a deoxyribose sugar, a phosphate group, and a nitrogenous base. True False Feedback: True A deoxyribose sugar with a nitrogenous base (and without a phosphate group) is a nucleoside. True False Feedback: True For the DNA sequence written as “A C G T”, the A is at the 3’ end, and the T is at the 5’ end. True False Feedback: False Adjacent nucleotides are linked through hydrogen bonds and the nitrogenous bases are linked through phosphodiester linkages. True False Feedback: False Hydrogen bond interactions occur between adenine and thymine (A and T), and between cytosine and guanine (C and G). ANTIBIOTIC RESISTANCE LAB|MICR 290 M04 PAGE 7 MODULE 04 COMPANION GUIDE MICR 290 True False Feedback: True DNA is a polymer of nucleosides. True False Feedback: False If you are unsure about any of the concepts in this activity, make sure to review the content in this section before progressing through the remainder of this module. In this section, you reviewed the chemical structure of DNA. You also explored the covalent and non-covalent bonds that link the different components of DNA. This includes phosphodiester bonds, which connect adjacent nucleotides, and hydrogen bonds, which bind two strands of DN A together to form a double helix. You reviewed the fact that DNA is typically double-stranded, forming an antiparallel helix, and that the directionality of DNA is important for replication and transcription. References: 1. Atdbio. (n.d.). Figure 4. Structures of a deoxynucleoside and a deoxynucleotide. Retrieved 1 September 2020, from https://www.atdbio.com/content/5/Nucleic-acid- structure. 2. TheFreeDictionary.Com. (n.d.) Full Size Picture deoxyribonucleic-acid.jpg. Retrieved September 16, 2020, from http://medical- dictionary.thefreedictionary.com/_/viewer.aspx?path=dorland&name=deoxyribonucl eic-acid.jpg 3. LJNovaScotia. (n.d.) Dna Biology Science Free Photo. Retrieved 1 September 2020, from https://www.needpix.com/photo/925213/dna-biology-science-molecule- genetic-gene-medical-structure-scientific. 4. VHV. RS. (n.d.). Dna Chemical Structure Cropped - Dna Structure Dna Molecule Diagram, HD Png Download. Retrieved 1 September 2020, from https://www.vhv.rs/viewpic/hToJRRR_dna-chemical-structure-cropped-dna- structure-dna-molecule/. End of Section 01 ANTIBIOTIC RESISTANCE LAB|MICR 290 M04 PAGE 8 MODULE 04 COMPANION GUIDE MICR 290 SECTION 02: POLYMERASE CHAIN REACTION The polymerase chain reaction (PCR) is an extremely important technique for amplifying DNA, allowing the creation of a massive number of copies of a specific nucleotide sequence. PCR is often used for diagnostic purposes. For example, PCR-based assays are used to identify SARS CoV-2, the virus behind the COVID-19 pandemic, amplifying small amounts of viral genetic material from patient samples so that they can be detected using additional lab techniques. PCR is also a vital tool for biotechnology research, where it is used to synthesize DNA strands which are used in down-stream applications (e.g. for preparing new plasmids). PCR Components PCR is used to synthesize double-stranded DNA. To set up PCR in the lab, several components must be mixed together in order to amplify a sequence of DNA. You will learn about each of these components in this section. DNA Polymerase and Deoxynucleoside Triphosphates (d N T Ps) The enzyme DNA polymerase is used to synthesize double-stranded DNA, adding nucleotides to the 3ʹ end of a growing DNA strand. These nucleotides are derived from deoxynucleoside triphosphates (d N T Ps), the building blocks of DNA synthesis. There are four d N T Ps: d A T P, d C T P, d G T P, and d T T P. Chemically, DNA polymerase forms a covalent bond between the hydroxyl group (O H) on carbon 3 of a deoxyribose sugar (located at the 3ʹ end of a DNA strand) and the phosphate group attached to carbon 5 of the d N T P, releasing pyrophosphate. DNA polymerase reads the nucleotide sequence of a strand of template DNA, and adds complementary nucleotides to the 3ʹ end of a primer, a short single- stranded molecule of DNA that binds to the template DNA. DNA polymerase forms a covalent bond between the O H group at the 3’ end of the primer and the phosphate group of the d N T P, releasing pyrophosphate.1 Template DNA ANTIBIOTIC RESISTANCE LAB|MICR 290 M04 PAGE 9 MODULE 04 COMPANION GUIDE MICR 290 DNA polymerase needs a template in order to determine which of the four d N T Ps to incorporate into the 3ʹ end of a growing strand of DNA. This enzyme does so by reading the strand of DNA that is complementary to the strand that is being synthesized. So, as DNA polymerase synthesizes DNA at the 3ʹ end of the growing strand, it reads the antiparallel strand (which is oriented from 3ʹ to 5ʹ) to determine which nucleotides to incorporate. Without this template, DNA polymerase cannot synthesize new DNA. DNA polymerase uses the template DNA strand to determine which nucleotides to add to the growing D NA strand.1 In summary: 1. DNA polymerase extends the 3ʹ end of a growing strand of DNA, and 2. DNA polymerase needs a complementary strand of template DNA in order to synthesize DNA. Take a moment to think about the question before clicking the button to continue. Most cellular DNA is double-stranded and does not have exposed 3ʹ ends that can be extended by DNA polymerase. How can DNA polymerase amplify a particular DNA sequence during PCR? Feedback: Dr. Lohans’ Feedback: This is addressed in PCR by using oligonucleotide primers, which are short single-stranded DNA molecules. Oligonucleotide Primers Primers (or oligonucleotides, or oligos) for PCR are single-stranded DNA molecules that are typically 15-25 nucleotides in length. A typical PCR experiment requires two primers, one complementary to each strand of the template DNA. The nucleotide sequences of the primers, which can be chemically synthesized, are designed to be complementary to the nucleotide sequence of the template DNA. If single-stranded primers are added to the double-stranded template DNA under the appropriate conditions, the primers bind to the complementary region of the template DNA. This satisfies the requirements for DNA polymerase ANTIBIOTIC RESISTANCE LAB|MICR 290 M04 PAGE 10 MODULE 04 COMPANION GUIDE MICR 290 mentioned above: DNA polymerase can add nucleotides to the 3ʹ end of the primer molecule, and it uses the complementary strand of the template DNA in order to determine which nucleotides to add. The nucleotide sequences of the primers determine which part of the template DNA is amplified. Oligonucleotide primers are complementary to the template DNA and are used to amplify a particular D NA sequence in PCR.1 Activity: Using your knowledge about the components of PCR, match each component to its function in PCR. Choices: DNA sample that contains the sequence to be amplified. Short single-stranded DNA molecules, usually consisting of 15-25 nucleotides. Enzyme that synthesizes new DNA, adding nucleotides to the 3’ end of a growing DNA strand. Building blocks for DNA synthesis used by DNA polymerase. DNA Polymerase Oligonucleotide Primers Deoxynucleoside Triphosphates (d N T Ps) Template DNA Feedback: Template DNA DNA sample that contains the sequence to be amplified. Oligonucleotide Primers Short single-stranded DNA molecules, usually consisting of 15-25 nucleotides. DNA Polymerase Enzyme that synthesizes new DNA, adding nucleotides to the 3’ end of a growing DNA strand. Deoxynucleoside Triphosphates (d N T Ps) Building blocks for DNA synthesis used by DNA polymerase. Take a moment to think about this question before clicking to listen to Dr. Lohans’ response. If you combine all the ingredients of PCR (template DNA, primers, DNA polymerase, and d N T Ps) ANTIBIOTIC RESISTANCE LAB|MICR 290 M04 PAGE 11 MODULE 04 COMPANION GUIDE MICR 290 and incubate the mixture at room temperature, nothing will happen - why not? Feedback: Listen to Dr. Lohans’ response. Start of Audio Transcript: The template for PCR is (usually) a long double-stranded molecule of DNA. The two strands of DNA stick together strongly due to the hydrogen bond interactions formed between complementary nucleotides. At room temperature, these two strands will mostly stay stuck together, which prevents the primers from binding. Therefore, DNA polymerase is not able to extend the 3ʹ end of the primers. In addition, although the primers are complementary to the template DNA, they are much shorter in length than the template DNA. The amount of favourable base pair interactions formed between the primer and template cannot compete with the favourable base pair interactions formed between the two longer strands of template DNA. Therefore, at room temperature, it is unfavourable for the primer to bind to the template. This can be overcome by adjusting the temperature of the reaction. End of Audio Transcript. PCR Temperatures As you can now appreciate, template DNA is (usually) double-stranded, with base pair interactions holding the two complementary DNA strands together (e.g. hydrogen bonds between A and T, and between C and G). The template DNA for PCR ranges from thousands (or kilobases, kb) to millions (or megabases, Mb) of nucleotides in length. Thus, the two strands of the template are tightly held together. To separate the template DNA strands, the temperature of the PCR sample must be adjusted. The temperature programme used for PCR consists of three main steps: melting, annealing, and extension. Learn the significance of the different temperatures in the PCR process. Melting Temperature (>90 °C) To overcome the strong association between both strands of the DNA template, the temperature of the sample must be increased, separating (denaturing, or melting) the double-stranded DNA into two separate strands. The temperature at which this occurs is the melting temperature. Samples for PCR are incubated at >90 °C to melt the double-stranded template DNA. The denaturation/melting step separates double-stranded DNA into two single strands of DNA. Annealing Temperature (50 – 65 °C) ANTIBIOTIC RESISTANCE LAB|MICR 290 M04 PAGE 12 MODULE 04 COMPANION GUIDE MICR 290 In order for the primers to bind to the template, the PCR temperature must be decreased. The temperature is lowered to the point at which the primers bind (or anneal) to the template; this is known as the annealing temperature (usually 50 – 65 °C). The specific annealing temperature is important. If it is too high, the primers are unable to bind to the template. However, if it is too low, the primers may bind non-specifically to other parts of the template, resulting in non-specific DNA amplification (because the primer binding sites determine what nucleotide sequence is amplified). The optimal annealing temperature will depend on the nucleotide sequence of the primers, and must be determined case-by-case. The annealing step allows the two primers to bind to the template DNA. The nucleotide sequences of the primers are complementary to the sequences of the template DNA strands. Extension Temperature (68 - 72 °C) After the annealing step, the primers are bound to the template. Then, the temperature of the PCR mixture is raised to the optimal temperature for the DNA polymerase, known as the extension temperature (~72 °C). At this temperature, the DNA polymerase adds d N T Ps to the 3' ends of the annealed primers. Special thermostable DNA polymerases that are active at high temperatures are used for PCR. You will learn more about these polymerases later in this section. The extension step allows the primers to be extended by DNA polymerase. Note: In the annealing step, one primer is complementary to the top strand of template DNA, and the other primer is complementary to the bottom strand. This means that the top primer has the same nucleotide sequence as the corresponding region of the bottom template DNA strand, and vice versa for the bottom primer and top template strand. Thermostable DNA Polymerases Most DNA polymerase enzymes function optimally at lower temperatures. For example, enzymes from Escherichia coli (E. coli) typically work best at 37 °C, and would unfold (denature) at the temperatures needed for PCR. To overcome this, PCR uses a thermostable DNA polymerase produced by thermophilic bacteria. These enzymes have evolved so that they can function at high temperatures ANTIBIOTIC RESISTANCE LAB|MICR 290 M04 PAGE 13 MODULE 04 COMPANION GUIDE MICR 290 and will not denature during PCR. Taq polymerase from the bacterium Thermus aquaticus is a thermostable DNA polymerase that is commonly used for PCR. Timing of PCR Temperature Steps Clearly, choosing the correct temperatures is critical for PCR to work properly. The length of time a PCR sample spends at each temperature is also very important. The PCR sample must be incubated at the melting temperature for long enough to allow for the double-stranded template DNA to melt into single DNA strands. Then, the sample must be incubated at the annealing temperature for long enough to allow for primers to bind to the template (but not for too long, or the template will re-form double- stranded DNA). The timing of the denaturing and annealing steps does not typically need to be adjusted between different experiments. The length of time that the PCR sample is incubated at the extension temperature (i.e. the extension time) depends on the length of the DNA sequence to be amplified. For the amplification of shorter sequences of DNA (or amplicons), the extension time does not need to be very long. Conversely, for longer amplicons, DNA polymerase needs more time to complete the DNA synthesis. The extension time is often based on the number of kb (kilobases) of DNA to be synthesized; different DNA polymerases require different lengths to synthesize one kb of DNA. These three steps, melting, annealing, and extension, constitute a single cycle of PCR. The presented figure graphically represents a single cycle of PCR. Reveal the temperature and incubation time for each step of a single representative PCR cycle. Melting Step Temperature: 95 °C Incubation Time: 30 s Annealing Step Temperature: 65 °C Incubation Time: 30 s Extension Step Temperature: 72 °C Incubation Time: 60 s Additional Cycles of PCR So far, you have considered a single cycle of PCR, in which the template is denatured, primers anneal, and DNA polymerase extends the 3ʹ end of the primers. This synthesizes one copy of the amplicon for each molecule of template DNA present. What happens if the same sample is then treated to the same temperature cycle again? ANTIBIOTIC RESISTANCE LAB|MICR 290 M04 PAGE 14 MODULE 04 COMPANION GUIDE MICR 290 In a second cycle of denaturation, annealing, and extension, the primers will still anneal to the template DNA, and DNA polymerase will still extend the 3ʹ end of the primers. However, primers can also anneal to the DNA that was synthesized in the first cycle: the product of the first cycle can act as template DNA. The DNA product of the first PCR cycle can act as template DNA in the second PCR cycle. A temperature programme for PCR typically consists of 20-30 cycles, with each cycle consisting of denaturation, annealing, and extension steps. Instruments known as thermocyclers are used for these experiments, rapidly heating and cooling samples to achieve the different temperatures needed for PCR. To ensure efficient thermocycling, PCR is generally carried out with small sample volumes (10 - 50 uL) and in thin-walled tubes that allow for rapid heat transfer. In addition to these cycles of denaturation, annealing, and extension steps, PCR methods typically begin with a long denaturation step and end with a long extension step. The initial denaturation step ensures that all double-stranded DNA is separated into single strands, while the final extension step ensures that the synthesis of all DNA strands is complete. As the DNA synthesized during one cycle can act as the template in subsequent cycles, the amount of P CR product theoretically doubles in each cycle. As a consequence of this, PCR can exponentially amplify a DNA sequence. This explains the sensitivity of PCR as an analytical technique, as it can amplify even very small amounts of template DNA. The exponential increase in PCR product as the number of cycles increases. By cycle 25, the reaction is no longer exponential due to limited resources (e.g. the amount of DNA polymerase, remaining d N T Ps, etc.) ANTIBIOTIC RESISTANCE LAB|MICR 290 M04 PAGE 15 MODULE 04 COMPANION GUIDE MICR 290 Four cycles of PCR produce 16 copies of double-stranded DNA from just one stand of template DNA. By cycle 20, over one million copies would be produced. The size and location of the DNA sequence that is amplified during PCR is determined by the nucleotide sequences of the primers. Two primers must be used in order for PCR to work properly, with one primer complementary to each strand of the template DNA. In addition, the 3ʹ ends of the primers must be pointing towards each other when they are bound to the template DNA. The region between the two primer binding sites is the region that is amplified during PCR. So, for example, if two primers are designed to bind to different regions of the template DNA that are 1 kb apart (with one primer binding to each strand of template), the major PCR product will be 1 kb in size, corresponding to the sequence between the two primer binding sites. Take a moment to think about the answer to the question before listening to Dr. Lohans’ response. Why does the binding site of one primer impact the length of the DNA strand that is synthesized from the other primer? Feedback: Listen to Dr. Lohans’ response. Start of Audio Transcript: You might expect that the length of the DNA strand produced during PCR is based solely on the extension time. However, the positions on the template to which the primers bind determines the length of the PCR product. This results from the ability of PCR to use DNA produced in early cycles as the template for later cycles. Importantly, only the 3ʹ ends of the primers are extended by DNA polymerase, and not the 5ʹ ends. As the PCR progresses, most of the DNA synthesis uses the products from earlier cycles as a template. Because only the 3ʹ ends of primers are extended, the PCR-derived template does not contain the nucleotide sequences that are 5ʹ to the primer binding sites from the initial template. Therefore, the major PCR product results from amplification of the DNA sequence located between the two primer binding sites. Remember for DNA replication to occur, DNA polymerase requires a template strand and a free 3’ end. End of Audio Transcript. ANTIBIOTIC RESISTANCE LAB|MICR 290 M04 PAGE 16 MODULE 04 COMPANION GUIDE MICR 290 The ability of PCR products to act as a template limits the length of the DNA produced in the next cycle. Use what you have learned so far about PCR to answer the question. If you amplify a gene using PCR, how might you determine whether or not this experiment has worked properly? Feedback: Dr. Lohans’ Feedback: You can analyze the products of the PCR using agarose gel electrophoresis. By comparing the PCR products with a DNA ladder, you can determine: i) whether or not DNA amplification occurred based on whether or not a band is observed in the agarose gel, and, ii) if the amplified band is of the expected size, as compared to the DNA ladder. DNA Polymerase Fidelity There are a variety of different DNA polymerases available for carrying out PCR. Notably, these different enzymes differ in the fidelity with which they catalyze DNA synthesis. DNA polymerases may make mistakes, incorporating the wrong nucleotide during DNA synthesis. This can result in the presence of mutations in the PCR product. Furthermore, if the mistake occurs early on during the PCR protocol, the mutated DNA acts as the template for subsequent cycles, propagating the error. ANTIBIOTIC RESISTANCE LAB|MICR 290 M04 PAGE 17 MODULE 04 COMPANION GUIDE MICR 290 The impact of DNA polymerase incorporating an incorrect nucleotide. These mutations can be avoided by using high fidelity DNA polymerases that are less likely to incorporate incorrect nucleotides during DNA synthesis. In addition, some DNA polymerases have proofreading activity: these enzymes recognize if the wrong nucleotide has been incorporated into a growing strand of DNA, and are able to remove and fix the mutation. The proofreading ability of DNA polymerase limits the amount of mutations that occur during PCR. ANTIBIOTIC RESISTANCE LAB|MICR 290 M04 PAGE 18 MODULE 04 COMPANION GUIDE MICR 290 Primer Design Primers determine which section of the template DNA is amplified during PCR. Clearly, the proper design of the primers is critical to the success of a PCR experiment, ensuring that the P C R product is the necessary size and sequence. To illustrate the process of designing primers, consider the following sequence of DNA. If you like, you can copy the given sequences over into your notes or a text editor so that you may manipulate the text. This may be helpful when completing the activities in this module. 5ʹ-A T G A C T G A T C G A T C G A T T C G A A C G T C G A C G A T C G A A C G C A T G G A A T T T C G A-3ʹ Now, suppose you wanted to use PCR to amplify the sequence that is highlighted: 5ʹ-A T G A C T G A T C G A T C G A T T C G A A C G T C G A C G A T C G A A C G C A T G G A A T T T C G A- 3ʹ You will need to design one primer that binds to this strand of DNA, and another primer that binds to the complementary strand. So, it is helpful to write out the sequence of the other strand of DNA. Answer the question using what you know about the structure of DNA and the nucleotide

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