MICR 290 Module 03 Companion Guide PDF

MICR 290 oiw ANTIBIOTIC RESISTANCE LAB MODULE 03 PURIFICATION ANALYSIS OF BACTERIAL D N A 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 03 COMPANION GUIDE MICR 290 TABLE OF CONTENTS SECTION 00: Module Overview............................................................................................................................ 3 SECTION 01: Bacterial DNA................................................................................................................................... 4 SECTION 02: DNA Purification............................................................................................................................19 SECTION 03: Analysis of DNA..............................................................................................................................29 SECTION 04: DNA Sequencing Data...................................................................................................................41 SECTION 05: Bioinformatics Approaches for DNA Sequence Analysis..........................................................45 ANTIBIOTIC RESISTANCE LAB|MICR 290 M03 PAGE 2 MODULE 03 COMPANION GUIDE MICR 290 SECTION 00: MODULE OVERVIEW This module will introduce you to the purification and analysis of bacterial DNA. You will learn how bacterial DNA is organized, how bacteria take up new DNA, and some of the laboratory techniques and methods used to purify and analyze DNA samples. Additionally, you will be introduced to two common bioinformatics tools and will practice using them with examples relevant to the laboratory setting. Watch the video of your instructor, Dr. Lohans, introducing the concepts that are covered in Module 03 (1:20). Visit the online module to view this video. After completing this module you will be able to: 1. Describe the major mechanisms by which bacteria take up DNA and explain how these mechanisms lead to the dissemination of antibiotic resistance. 2. Describe the major steps involved in spin column-based DNA purification. 3. Predict the outcome of agarose gel electrophoresis and Southern blot analysis, given information regarding the sample to be analyzed. 4. Interpret experimental results obtained from agarose gel electrophoresis and Southern blots, and apply this analysis to answer scientific questions. 5. Compare and contrast common techniques used for analyzing DNA samples and sequences. 6. Apply common bioinformatics tools (e.g. Translate, BLAST) in order to identify genes and proteins. ANTIBIOTIC RESISTANCE LAB|MICR 290 M03 PAGE 3 MODULE 03 COMPANION GUIDE MICR 290 SECTION 01: BACTERIAL D NA In the previous module, the central dogma of molecular biology was reviewed: DNA is transcribed to RNA, and RNA is translated into proteins. Before moving forward, it is important to review some additional molecular details in these processes before discussing the different types of genetic material found in bacteria, the mechanisms by which bacterial DNA can be changed, and the various methods by which bacterial cells can acquire new DNA. DNA is a polymer made up of the nucleotides: adenosine (A), cytidine (C), guanosine (G), and thymidine (T). The regions of DNA that encode for proteins are known as genes. Genes are composed of codons, groupings of three nucleotides. Genes begin with a start codon (nucleotide sequence: A T G) and end with a stop codon (nucleotide sequence: TAG, T A A, or T G A). Between the start codon and stop codon are a series of codons that encode for the amino acids that make up a protein. Schematic showing the structure of a typical gene1. RNA is also a polymer, and is made up of the nucleotides: adenosine (A), cytidine (C), guanosine (G), and uridine (U); note that U is used in place of T. RNA polymerase recognizes a DNA sequence located before the gene known as a promoter, and synthesizes an mRNA transcript of the gene. This transcript is translated by the ribosome in order to produce a protein. The ribosome recognizes the codons in the mRNA transcript, initiating protein synthesis at the start codon, selecting which amino acid to incorporate based on the codons, and terminating protein synthesis at the stop codon. DNA is transcribed into mRNA by the enzyme, RNA polymerase. The mRNA is then translated by ribosomes into the amino acid sequence of a protein. Bacterial DNA Several different kinds of DNA molecules can exist in a bacterial cell. ANTIBIOTIC RESISTANCE LAB|MICR 290 M03 PAGE 4 MODULE 03 COMPANION GUIDE MICR 290 1. Chromosome: The major DNA component in a bacterial cell is the chromosome, which normally occurs as a double-stranded, circular molecule. 2. Plasmid: In addition to the chromosome, bacteria often contain plasmids, which are mobile genetic elements that can be transferred between bacterial cells. 3. Transposons: Bacteria also contain transposons, another type of mobile genetic element. You will now delve into these different forms of genetic material in bacteria. Chromosome The bacterial chromosome is typically several megabase pairs (M b, which is one million base pairs) in size, with each strand of DNA consisting of several million nucleotides. While the genetic material of eukaryotes is located in a membrane-bound nucleus, the bacterial chromosome is located in a region of the cytoplasm known as the nucleoid. The chromosome encodes for all of the essential functions for bacterial growth, metabolism, and replication. This includes genes that encode for proteins and sequences that encode for ribosomal RNA (rRNA) and transfer RNA (tRNA). Genes that encode for proteins with related functions tend to be grouped together on the bacterial chromosome, organized into structures known as operons. For example, metabolism of a particular sugar may require several enzymatic steps; the genes that encode for the enzymes responsible for these steps are often clustered together into an operon. The genes in an operon are often transcribed together as a long strand of mRNA. This single strand of mRNA can be translated by the ribosome to produce all of the different proteins encoded in the operon. An advantage of this setup is that the transcription of these related genes can all be regulated by a single promoter and operator. In bacterial cells, genes of related function are often organized together in an operon and transcribed under the control of a single promoter2. Plasmids ANTIBIOTIC RESISTANCE LAB|MICR 290 M03 PAGE 5 MODULE 03 COMPANION GUIDE MICR 290 Through the process of binary fission, a bacterium replicates its chromosome and undergoes further growth before dividing into two identical cells3. Leaving Cert Biology When bacterial cells replicate by binary fission, the two offspring cells each inherit a copy of the chromosome. This transfer of genetic material from parent cells to their offspring is known as vertical gene transfer. This differs from horizontal gene transfer, processes by which bacteria acquire DNA molecules from external sources. Diagram of bacterial cell which includes both chromosomal and plasmid DNA, and a transmission electron micrograph (TEM) of plasmid DNA4. Plasmids are circular, double-stranded molecules of DNA that are found in the bacterial cytoplasm. They tend to be smaller than the chromosome, ranging in size from 1kilobase pair (k b, which is a thousand base pairs) to more than 100 k b. A single bacterial cell can contain several different plasmids, multiple copies of the same plasmid, or no plasmids at all. ANTIBIOTIC RESISTANCE LAB|MICR 290 M03 PAGE 6 MODULE 03 COMPANION GUIDE MICR 290 Plasmids replicate independently from the bacterial chromosome. Some plasmids encode the elements (e.g. genes) required for their own replication, while other plasmids rely on elements encoded by the chromosome. The number of copies of a particular plasmid in a single bacterial cell is known as its copy number, which is determined by specific interactions between the plasmid and the DNA replication enzymes in the cell. For a plasmid with a low copy number, only a few copies of this plasmid will exist in each cell. By contrast, a bacterial cell can contain hundreds to thousands of copies of a high copy plasmid. While the chromosome encodes all of the proteins involved in the core processes required during the bacterial lifespan, plasmids encode functions that are considered to be non-essential. However, the genes encoded by plasmids can be extremely beneficial to a bacterial cell. In fact, plasmid-encoded genes can be essential for bacterial survival under certain conditions. The distinction is that these genes are not essential under all conditions. As an example, many plasmids carry genes responsible for antibiotic resistance. Under normal circumstances, such plasmids are not essential for a cell. However, in the presence of an antibiotic, they can be vital for survival. Environmental conditions can exert a selective pressure that favour bacteria that contain such plasmids. In the absence of an antibiotic, replication of the plasmid may be too energetically demanding and may slow the growth of bacteria. Hence, under normal circumstances, bacteria which lack the plasmid may be selected for. The functions of genes encoded by a plasmid typically fall into one of five general classes. Learn about the five classes of plasmids. Fertility Factors Fertility factors (also known as F-plasmids or F factors) encode the necessary functions for bacteria to undergo conjugation, a type of horizontal gene transfer in which a copy of the plasmid is transmitted from one bacterial cell to another. Resistance Plasmids Resistance plasmids (or R factors) contain genes that confer bacteria with resistance to certain antibiotics. Some resistance plasmids contain several different resistance genes, conferring resistance to many different classes of antibiotics. Virulence Plasmids Virulence plasmids contain genes that make bacteria pathogenic. For example, some virulence plasmids cause bacterial cells to produce a capsule, a virulence factor described in Module 02. Degradative Plasmids Degradative plasmids expand the metabolic scope of a bacterium, allowing it to catabolize (digest) a broader range of substances. Col Plasmids ANTIBIOTIC RESISTANCE LAB|MICR 290 M03 PAGE 7 MODULE 03 COMPANION GUIDE MICR 290 Col plasmids contain genes involved in the production of bacteriocins, which are peptides or proteins that bacteria produce in order to kill other bacteria. Many plasmids can be transferred between different bacterial cells through horizontal gene transfer mechanisms. As such, they are a type of mobile genetic element. Transposons Transposons are short sequences of DNA that are capable of moving between different strands of DNA. They can jump from a chromosome to a plasmid, from a plasmid to a chromosome, or even to different positions within a chromosome or plasmid. Due to this ability, transposons are another type of mobile genetic element. Transposons are relatively small, with many containing only a single gene. The movement of transposons between DNA sequences is referred to as transposition. Simplified mechanism of how a transposon jumps and inserts into a new DNA sequence5. Note: Some transposons also contain an antibiotic resistance gene, contributing to the spread of antibiotic resistance. For example, if an antibiotic-susceptible bacterial cell takes up a strand of DNA that contains a transposon, this transposon could insert into the chromosome of this cell, thereby conferring the cell with antibiotic resistance. Answer the question using what you have learned about the types of bacterial DNA. Which types of bacterial DNA are paired with the correct function? Select all that apply. a) Transposons – short sequences of DNA that are capable of moving between different strands of DNA. b) R Factor – contains genes that confer bacteria with resistance to certain antibiotics. c) F Factor – expands the metabolic scope of a bacterium, allowing it to catabolize (digest) a broader range of substances. d) Chromosome – encodes the necessary functions for bacteria to undergo conjugation. e) Col Plasmid – causes bacterial cells to produce a capsule. f) eVirulence Plasmid – contains genes that make bacteria pathogenic. g) Degradative Plasmid - contains genes involved in the production of bacteriocins. Feedback: ANTIBIOTIC RESISTANCE LAB|MICR 290 M03 PAGE 8 MODULE 03 COMPANION GUIDE MICR 290 The correct pairs are a, b, and f. F factors encode the necessary functions for bacteria to undergo conjugation, not the chromosome. Col plasmids contain genes involved in the production of bacteriocins and degradative plasmids expand the metabolic scope of a bacterium. Mechanisms of Bacterial Evolution Changes to the DNA in a bacterial cell (i.e. its genotype) impact the behaviour and function of this cell (i.e. its phenotype). These changes can have significant medical consequences, impacting the pathogenicity and antibiotic susceptibility of a bacterial cell (pathogenicity in this context refers to the ability to cause disease). Changes to DNA represent the basis of natural selection, where changes that improve survival are more likely to be passed onto offspring. So, how does this happen in bacteria? You will first consider mutations and recombination, mechanisms by which DNA sequences can be changed. Then you will learn about conjugation, transformation, and transduction, which are horizontal gene transfer mechanisms by which bacteria can acquire new DNA sequences. Mutations Mutations are permanent changes to the sequence of a DNA molecule, caused by mutagens such as chemicals and radiation, or when mistakes are made during DNA replication. There are several different kinds of mutations. Base substitutions are common mutations in which a nucleotide is substituted with one of the other three nucleotides (e.g. an adenosine is substituted with a cytidine). If this nucleotide is part of a gene that encodes for a protein, the mutation can impact the structure and function of that protein. ANTIBIOTIC RESISTANCE LAB|MICR 290 M03 PAGE 9 MODULE 03 COMPANION GUIDE MICR 290 Codon tables, such as this, represent how combinations of nucleotides map to the production of particular amino acids. To read the table, start to the left with the first base, then find the second base along the top. The box where these two meet shows all the possible combinations of codons with the third base, along with the amino acid or instructions specified by that particular codon 6. Learn about the three main types of base substitutions and their impacts. Missense Mutation The codon that encodes for a particular amino acid is mutated into a codon that encodes for a different amino acid. This results in the production of a protein with a different amino acid sequence. A missense mutation results in an amino acid substitution7. Nonsense Mutation The codon that encodes for a particular amino acid is mutated into a stop codon. The ribosome will recognize this new stop codon, and will stop translating before the full protein has been synthesized. The resulting protein will be truncated. ANTIBIOTIC RESISTANCE LAB|MICR 290 M03 PAGE 10 MODULE 03 COMPANION GUIDE MICR 290 A nonsense mutation results in the formation of a stop codon7. Silent Mutation The codon that encodes for a particular amino acid is mutated into another codon that encodes for the same amino acid. For example, the codon A A A encodes for lysine. If this codon is mutated to A A G, it will still encode for lysine. Therefore, silent mutations do not change the protein encoded by a gene. A silent mutation has no effect on the protein sequence7. In frameshift mutations, a gene sequence may lose a nucleotide or gain a nucleotide. Ribosomes interpret mRNA in a particular reading frame, based on the sequence of codons that make up a gene. If nucleotides are added to or removed from this sequence, the reading frame will be disrupted. This will disrupt the ability of the ribosome to read the codons that occur after the mutation and may completely change the amino acid sequence encoded by the gene. Deletion of nucleotides shifts the mRNA reading frame, and disrupts the amino acid sequence7. Answer the questions using what you have learned about the types of mutations. 1 of 2: What would happen to the reading frame if a gene gained three additional adjacent nucleotides as the result of a frameshift mutation? Dr. Lohans’ Response: Assuming the adjacent nucleotides do not code for a stop codon, an additional amino acid would be incorporated into the protein encoded by the gene. In addition, depending on where the new nucleotides are inserted, one of the other amino acids may be substituted with a different amino acid. However, the reading frame would not be disrupted. All of the codons that follow the frameshift mutation would still encode for the same amino acids as they did originally. ANTIBIOTIC RESISTANCE LAB|MICR 290 M03 PAGE 11 MODULE 03 COMPANION GUIDE MICR 290 Nonetheless, the addition of even just a single amino acid to a protein could disrupt its structure and activity. 2 of 2: Which is the correct order of types of mutations ranked from the greatest to the least impact on protein function? a) Silent mutations, missense mutations, nonsense mutations, frameshift mutations. b) Frameshift mutations, nonsense mutations, missense mutations, silent mutations. c) Silent mutations, nonsense mutation, missense mutations, frameshift mutations. d) Frameshift mutations, missense mutations, nonsense mutations, silent mutations. Feedback: b). Silent mutations, which do not result in a change in amino acid sequence, have no effect on protein function. Depending on the role of the amino acid being substituted, and depending on what amino acid it is substituted with, missense mutations often have little effect on protein activity. However, some missense mutations can cause a complete loss of activity. Nonsense mutations and frameshift mutations tend to be far more detrimental to protein function, either preventing the complete protein from being produced by the ribosome, or significantly altering the amino acid sequence of the protein. Mutations can have a wide range of effects on the survival and behaviour of bacterial cells. Learn about how mutations can be deleterious or beneficial. Deleterious Mutations Mutations to genes that encode for proteins that are essential for bacterial function can be lethal, preventing the mutated bacteria from replicating, and preventing this mutation from being propagated. Some mutations can disfavour bacterial growth without being lethal, while others are neutral and do not impact growth one way or another. Beneficial Mutations Some mutations are beneficial, giving the mutated bacteria a survival advantage over other bacteria in the environment. For example, if a bacteria mutates such that it is more resistant to an antibiotic, it will replicate more rapidly than other bacteria in the presence of the antibiotic. As a consequence, the mutated bacteria will pass on the resistance mutation to its offspring. The selective pressure exerted by the antibiotic selects for bacteria that have this mutation. The impact of a mutation depends on many factors. While a mutation may provide a bacterial strain with a growth advantage in the presence of antibiotic, this same mutation may hinder growth in the absence of antibiotic. Recombination Recombination is another mechanism by which DNA sequences can be changed in bacteria. Unlike mutations, which typically impact a small number of nucleotides, recombination involves the exchange of longer sequences of nucleotides (e.g. genes) between two molecules of DNA. Bacteria are capable of taking in foreign DNA through several different mechanisms (described later in this section). If the ANTIBIOTIC RESISTANCE LAB|MICR 290 M03 PAGE 12 MODULE 03 COMPANION GUIDE MICR 290 nucleotide sequence of this foreign DNA is similar enough to the DNA present in the bacterial cell, recombination can occur. This process involves the incorporation of the foreign DNA into the bacterial chromosome (or plasmid). This process is typically catalyzed by enzymes known as recombinases. Diagram depicting recombination, in which foreign DNA is integrated into the bacterial chromosome. The foreign DNA may be from the environment, other bacteria, or a virus. Similar to mutations, recombination can have beneficial, neutral, and deleterious effects on a bacterial cell. Those genetic changes that benefit a bacterial cell will increase the likelihood of these changes being passed onto offspring cells through vertical gene transfer. This can lead to the emergence and spread of antibiotic resistance. Horizontal Gene Transfer There are three main mechanisms by which horizontal gene transfer can occur in prokaryotes: conjugation, transformation, and transduction. You will learn about each one in more detail throughout the rest of this section. ANTIBIOTIC RESISTANCE LAB|MICR 290 M03 PAGE 13 MODULE 03 COMPANION GUIDE MICR 290 In transformation, the cell takes up DNA directly from the environment. In conjugation, DNA, in the form of a plasmid, is transferred between cells. In transduction, a bacteriophage injects DNA into the bacterial cell8. Conjugation Conjugation is a mechanism of horizontal gene transfer by which genetic material is directly transferred from one bacterium to another. In Escherichia coli (E. coli), where these processes have been most well studied, bacterial conjugation typically requires a plasmid known as the fertility factor (or F factor, or F plasmid). This plasmid carries genes that encode for a sex pilus, a structure on the surface of a bacterial cell that forms a channel to another bacterial cell. Bacteria that contain the F factor plasmid are said to be F⁺, while those that do not are F⁻. Learn the steps of a typical conjugation process. Transfer of the F plasmid from an F + cell to an F - cell through conjugation9. 1 – Cell Attachment The sex pilus of a F+ cell (the donor) attaches to a F- cell (the recipient). The pilus contracts, drawing the cells together and forming a channel between them. 2 – DNA Transfer A single-strand of the F factor plasmid DNA is transmitted from the donor cell to the recipient cell. ANTIBIOTIC RESISTANCE LAB|MICR 290 M03 PAGE 14 MODULE 03 COMPANION GUIDE MICR 290 3 – DNA Synthesis The complementary strands of the plasmids are then synthesized in both cells, such that both cells have double-stranded F factor plasmids. As a result of this transfer, the F - cell becomes F+. Transformation Transformation is the process by which bacteria take up DNA from the environment. However, not all bacteria are capable of undergoing transformation; this depends on the competence of the bacteria. Some species of bacteria are naturally competent, meaning that they are naturally able to take up DN A from the environment. Other bacteria can be made artificially competent in the lab. Although E. coli is used extensively in the lab for manipulating DNA, it is not naturally competent. There are many established lab protocols for preparing competent E. coli cells, which can be easily transformed with plasmid DNA. Compare the transformation of plasmid and non-plasmid DNA. Transformation of Plasmid DNA Transformation of a bacterium with a plasmid is relatively simple: the plasmid passes through the bacterial cell envelope into the cytoplasm, where it is then replicated by bacterial enzymes. If the plasmid is beneficial to the bacterial cell, those bacteria that contain the plasmid will be favoured relative to bacteria that do not. Transformation of Non-Plasmid DNA Transformation is more complicated for non-plasmid DNA, and often requires several more steps. After foreign DNA binds to the surface of a bacterial cell, enzymes digest it into small pieces, and the fragments are transported into the cytoplasm. Once in the cytoplasm, many of the foreign strands of D NA will be digested by enzymes; however, there is a chance that some of this DNA will be incorporated into the bacterial chromosome (e.g. by recombination). Then, when the chromosome is replicated during bacterial reproduction, this foreign DNA will be passed onto the offspring of the bacterial cells. D NA that is not integrated into the chromosome is not replicated. Diagram depicting the transformation of a bacterial cell with DNA fragments. ANTIBIOTIC RESISTANCE LAB|MICR 290 M03 PAGE 15 MODULE 03 COMPANION GUIDE MICR 290 Transduction Transmission electron micrograph showing a bacteriophage on the surface of an Escherichia coli cell10 While conjugation involves bacteria acquiring DNA from other bacteria, and transformation involves bacteria acquiring DNA from the environment, transduction is the process by which bacteria acquire DN A from viruses. Viruses known as bacteriophages are infectious particles that contain genetic material. During the course of an infection, bacteriophages inject this genetic material into the cytoplasm of bacterial cells. Although these nucleic acids are typically copies of the viral genome, bacteriophages can also transfer other kinds of DNA (e.g. plasmids). Learn about the various steps of transduction. Various steps involved in the transduction of genetic material between a bacteriophage and bacterial cell. 1 – Infection Transduction begins with the infection of a bacterial cell with a bacteriophage, where the bacteriophage injects its genetic material into the bacterium. ANTIBIOTIC RESISTANCE LAB|MICR 290 M03 PAGE 16 MODULE 03 COMPANION GUIDE MICR 290 2 – Genome Replication The viral genetic material hijacks the cellular machinery of the bacterium, producing large numbers of copies of the bacteriophage genome and proteins. 3 – Capsid Assembly and Mispackaging The viral proteins assemble to form the three-dimensional capsid, which forms the “body” of the virus. Normally, the viral genetic material is packed into the capsid; however, DNA from the bacterial cell is sometimes packaged in the capsid instead. 4 – Bacterial Cell Lysis At this stage, the bacterial cell is lysed, releasing many new bacteriophages which will then go on to infect other bacterial cells. 5 – Transduction If a bacteriophage that contains bacterial DNA (instead of a viral genome) infects a new bacterial cell, this DNA will be injected into the cytoplasm of the new bacterium. 6 – Recombination At this stage, the foreign DNA can integrate into the bacterial chromosome (e.g. through recombination). The net result of this process is the transfer of DNA from one bacterium to another, with a bacteriophage acting as an intermediate. In this section you learned about the different forms of DNA that are found in bacteria. You also explored mobile genetic elements, such as plasmids and transposons, and saw how bacteria evolve through horizontal and vertical gene transfer. Now that you have taken a closer look at the theory, the rest of this module will explore experimental techniques used for working with DNA, beginning with DNA purification. Page Links: https://leavingcertbiology.net/chapter-20-kingdom-monera/ References: 1. Yourgenome. (n.d.). How do you identify the genes in a genome? Retrieved August 29, 2020, from https://www.yourgenome.org/facts/how-do-you-identify-the-genes-in-a-genome 2. Keenleyside, W. (2019). 12.7 Gene Regulation. Microbiology: Canadian Edition. Pressbooks. Retrieved August 29, 2020 from https://ecampusontario.pressbooks.pub/microbio/chapter/gene-regulation-operon- theory/ 3. Leaving Cert Biology. (2018). Chapter 20: Kingdom Monera. Retrieved August 29, 2020 from https://leavingcertbiology.net/chapter-20-kingdom-monera/ 4. Quora. (n.d.). What is the importance of plasmids in biotechnology? Retrieved August 29, 2020, from https://www.quora.com/What-is-the-importance-of-plasmids-in-biotechnology ANTIBIOTIC RESISTANCE LAB|MICR 290 M03 PAGE 17 MODULE 03 COMPANION GUIDE MICR 290 5. Rhoades, O. (2018) Transposons: Your D N A that’s on the go. Science in the News. Retrieved August 29, 2020, from http://sitn.hms.harvard.edu/flash/2018/transposons-your-D N A-thats- on-the-go/ 6. Genomenon. (n.d.). Codon Charts - Codon Table Sheets. Retrieved August 31, 2020, from https://www.genomenon.com/codon-chart/ 7. Keenleyside, W. (2019). 12.5 Mutations. In Microbiology: Canadian Edition. Pressbooks. Retrieved August 29, 2020, from https://ecampusontario.pressbooks.pub/microbio/chapter/mutations/ 8. Stecher, B., Maier, L., & Hardt, W.-D. (2013). “Blooming” in the gut: How dysbiosis might contribute to pathogen evolution. Nature Reviews Microbiology, 11(4), 277–284. Retrieved August 29, 2020, from https://doi.org/10.1038/nrmicro2989 9. Keenleyside, W. (2019). 12.6 How Asexual Prokaryotes Achieve Genetic Diversity. In Microbiology: Canadian Edition. Pressbooks. Retrieved August 29, 2020, from https://ecampusontario.pressbooks.pub/microbio/chapter/how-asexual-prokaryotes- achieve-genetic-diversity/ 10. Howard, L. (2012) CIL:41123, Enterobacteria phage T4, Escherichia coli. Dartmouth EM Facility. Retrieved August 2020, from http://www.cellimagelibrary.org/images/41123 End of Section 01 ANTIBIOTIC RESISTANCE LAB|MICR 290 M03 PAGE 18 MODULE 03 COMPANION GUIDE MICR 290 SECTION 02: D NA PURIFICATION Many different research applications depend on the ability to purify DNA. For example, Sanger sequencing and next-generation sequencing methods (introduced in Module 02) depend on relatively pure DNA samples in order to work properly. However, in a bacterial cell, DNA is present in a complex mixture consisting of proteins, sugars, lipids, and many different metabolites. Approaches used to purify DNA typically rely on the different chemical properties of DNA, as compared to other kinds of macromolecules. This section will discuss four common approaches: Phenol-Chloroform Extraction Ethanol Precipitation Solid-Phase Extraction (Spin Columns) Gel Extraction Although these approaches can be used to purify DNA from many different sources, here we focus on their use with bacterial cells. Many of these protocols require that the bacterial cells be broken open (or lysed), such that the DNA and other contents of the cell are released into solution. Bacterial cells are commonly lysed by using detergents, sonication (sound waves), and alkaline (basic) solutions, amongst other methods. Phenol-Chloroform Extraction This is a liquid-liquid extraction, using a combination of water, phenol, and chloroform. Phenol and chloroform are hydrophobic (non-polar) organic solvents, while water is polar. In a mixture of these three solvents, two different phases (or layers) occur. As phenol and chloroform are both non-polar, they are miscible and form a single layer. However, water is immiscible with phenol and chloroform, and so it forms a separate layer. When a crude mixture containing DNA is added to a mixture of phenol, chloroform, and water, the different chemical properties of the macromolecules contained in this mixture will determine which layer they appear in. Lipids, which are non-polar, enter the hydrophobic phenol-chloroform layer. Proteins may be both polar and non-polar, and so they are enriched at the interface between the phenol-chloroform layer and the water layer. Under appropriate conditions, DNA preferentially enters the aqueous (water) layer. After separating the macromolecules in these different layers, the aqueous layer (which contains the purified DNA) is collected. However, care must be taken not to collect too much of the aqueous layer, to avoid collecting the proteins located at the layer interface. View the contents of each layer. The layers of a phenol-chloroform extraction. Aqueous Phase ANTIBIOTIC RESISTANCE LAB|MICR 290 M03 PAGE 19 MODULE 03 COMPANION GUIDE MICR 290 Water Purified DNA Interface Proteins Organic Phase Phenol Chloroform Lipids Using what you have learned about phenol-chloroform extraction and the diagram provided, sort the items into the correct category. Chloroform Organic Phase Proteins Aqueous Phase Water Purified DNA Interface Lipids Phenol Feedback: Top Layer Middle Layer Bottom Layer Aqueous Phase Proteins Chloroform Water Interface Organic Phase Purified DNA Lipids Phenol Ethanol Precipitation Similar to phenol-chloroform extraction, ethanol precipitation is based on the poor solubility of DNA in organic solvents relative to other macromolecules. ANTIBIOTIC RESISTANCE LAB|MICR 290 M03 PAGE 20 MODULE 03 COMPANION GUIDE MICR 290 In this protocol, cold ethanol (or isopropanol) and a concentrated salt solution (such as sodium acetate) are added to the crude DNA sample that is dissolved in water. The solubility of the DNA in the solution is decreased by the sodium acetate salt. The positively charged sodium ions interact with the negatively charged phosphate backbone of DNA, neutralizing their charges. The ethanol in this mixture changes the properties of the solution, making the sodium ions interact more strongly with the DNA. As a consequence of the combined effects of the ethanol and sodium acetate, the DNA will precipitate, leaving most other macromolecules dissolved in solution. This mixture can then be centrifuged, pelleting the precipitated DNA. The supernatant, or liquid overlying the precipitate, is then carefully removed, thereby removing the dissolved contaminants from the insoluble DNA pellet. The pellet can then be gently washed with a cold ethanol-water solution to remove even more contaminants (including the excess sodium acetate), re-centrifuged, and the supernatant is carefully removed. The D NA pellet is then dried, removing all of the residual ethanol from the sample. At this point, the DNA can be re-dissolved in water or buffer and used for further experiments. View the contents of the sample at each step of ethanol precipitation. 1 – Starting Solution Crude DNA Sample Water DNA Proteins 2 – Precipitated Solution Precipitation Mixture Water Ethanol Sodium Acetate Proteins Precipitated DNA ANTIBIOTIC RESISTANCE LAB|MICR 290 M03 PAGE 21 MODULE 03 COMPANION GUIDE MICR 290 3 – Centrifuged Solution Supernatant Water Ethanol Sodium Acetate Proteins Pellet DNA Excess Sodium Acetate Remaining Contaminants 4 – Washed Pellet Mixture Washing Solution Cold Ethanol-Water Mixture Excess Sodium Acetate Remaining Contaminants Pellet DNA 5 – Solvent Removal Pellet Purified DNA 6 – Final Solution Purified DNA Sample Water or Buffer Purified DNA Solid-Phase Extraction (Spin Columns) Like the previous two methods, solid-phase extraction methods for DNA purification are based on the specific chemical properties of DNA. Solid-phase extraction involves the selective binding of DNA to a solid material/matrix, allowing other molecules to be washed away. This approach is widely used, and many kits are commercially available to streamline this protocol. When carried out on a small scale, these are known as minipreps. The most common solid phase used for DNA purification is silica, a material composed of silicon and oxygen (chemically similar to sand). Under certain conditions, DNA binds to silica while other ANTIBIOTIC RESISTANCE LAB|MICR 290 M03 PAGE 22 MODULE 03 COMPANION GUIDE MICR 290 macromolecules do not. These conditions include the use of ethanol, which decreases the solubility of DNA in solution, and chaotropic salts, which interfere with the hydrogen bond interactions between D NA and water, making the DNA interact more strongly with the silica. Although DNA binds selectively to silica, there must then be a way to remove other molecules from the sample. This is accomplished by using spin columns, small tubes fitted with a permeable silica membrane at the bottom. Spin columns are placed into collection tubes which can be centrifuged. When a solution is added to the top of a spin column and centrifuged, the solution will pass through the silica membrane and enter the collection tube. If ethanol or chaotropic salts are present, DNA in the solution will stick to the silica membrane, while all of the other molecules pass into the collection tube. Then, if water or a buffer is added to the spin column, the purified DNA can be eluted from the silica membrane into an empty collection tube. A spin column fitted with a silica membrane at the base. In the presence of ethanol and/or chaotropic salts, DNA binds to the silica membrane. Spin Column-Based Protocol Presented is a protocol for purifying plasmid DNA using spin columns. There are related protocols for other kinds of DNA purifications. Go through the steps of a typical spin-column protocol. Step 1: Resuspension ANTIBIOTIC RESISTANCE LAB|MICR 290 M03 PAGE 23 MODULE 03 COMPANION GUIDE MICR 290 A liquid bacterial culture is centrifuged, pelleting the bacterial cells. The cell pellet is then resuspended in a resuspension buffer. Step 2: Lysis Bacterial cells are broken open, typically by using an alkaline lysis buffer. This buffer may contain sodium dodecyl sulfate (S D S, a common detergent) and sodium hydroxide (NaOH, a strong base). The detergent disrupts the bacterial cell membrane(s), releasing their contents into solution. The alkaline conditions disrupt the hydrogen bonds that hold double-stranded DNA together, separating it into single strands; this is known as denaturation. Step 3: Neutralization A neutralization buffer is added which decreases the alkalinity of the solution, allowing hydrogen bonds to re-form between DNA strands. These conditions are designed so that shorter sequences of single-stranded DNA (like plasmids) are able to recombine to form double-stranded DNA (also known as annealing or renaturation). However, under these conditions, longer DNA molecules (such as chromosomal DNA) are not able to renature. As a result, single-stranded chromosomal DNA will stick to the denatured (unfolded) proteins in the mixture, forming an insoluble precipitate. In addition, the neutralization buffer contains chaotropic salts which will promote the binding of DNA to silica later in this protocol. Step 4: Centrifugation ANTIBIOTIC RESISTANCE LAB|MICR 290 M03 PAGE 24 MODULE 03 COMPANION GUIDE MICR 290 The neutralized mixture is centrifuged at a high speed. The renatured plasmid DNA stays in the supernatant, while the insoluble precipitate formed from proteins and chromosomal DNA is pelleted on the bottom of the tube. Step 5: Spin Column Loading The supernatant from the last step is loaded onto a spin column in a collection tube, and centrifuged. Due to the chaotropic salts present in the solution, the plasmid DNA will bind to the silica membrane in the spin column, while the other components will pass through into the collection tube. The contents of the collection tube are discarded. Step 6: Spin Column Washing A 80% ethanol solution (80% ethanol, 20% buffer) is added to the spin column, and the spin column is centrifuged. This washes contaminants from the bound plasmid DNA, including some of the chaotropic salts. The ethanol in this solution ensures that the DNA stays bound to the spin column. The wash solution that passes through the membrane into the collection tube is discarded. ANTIBIOTIC RESISTANCE LAB|MICR 290 M03 PAGE 25 MODULE 03 COMPANION GUIDE MICR 290 Step 7: Spin Column Elution Water or buffer is added to the spin column, and the spin column is centrifuged. In the absence of ethanol or chaotropic salts, the plasmid DNA no longer sticks to the silica membrane, and so it is eluted into the collection tube. Watch a video on how to perform a solid phase extraction using a spin column to purify plasmid DNA from bacterial cells. This will help solidify your understanding of the principles involved in spin-based column protocols used for DNA purification (3:21). Monarch Plasmid Miniprep Kit Protocol – View on Youtube Match the results to the expected error using what you have learned about the solid phase extraction protocol. Choices: The protein and chromosomal DNA did not precipitate DNA is eluted with the contaminants The DNA is not extracted The supernatant contains all of the DNA in the sample DNA won’t bind to the silica Forget to add a neutralization buffer Forget to add chaotropic salts Add the neutralization buffer before the lysis buffer Use a lysis buffer that is not alkaline Use a 20% ethanol 80% buffer solution during the washing step Feedback: Forget to add a neutralization buffer The protein and chromosomal DNA did not precipitate Forget to add chaotropic salts DNA won’t bind to the silica Use a lysis buffer that is not alkaline The supernatant contains all of the DNA in the sample ANTIBIOTIC RESISTANCE LAB|MICR 290 M03 PAGE 26 MODULE 03 COMPANION GUIDE MICR 290 Use a 20% ethanol 80% buffer solution during DNA is eluted with the contaminants the washing step Add the neutralization buffer before the lysis The DNA is not extracted buffer Gel Extraction Electrophoresis is a technique which separates molecules based on their charge. Agarose gel electrophoresis can be used to separate DNA molecules according to their size by passing them through a block of agarose, a gel-like material. This process also separates DNA from other kinds of macromolecules. This technique is covered in more detail later in this module; for now, you will learn how DNA can be removed from an agarose gel by gel extraction. Similar to the previous section on purifying plasmid DNA, the gel extraction protocol also uses spin columns fitted with silica membranes. The process typically involves: 1. The section of the agarose gel that contains DNA is cut out using a scalpel. 2. A buffer (containing chaotropic salts and/or ethanol/isopropanol) is added to the agarose slice, and the mixture is heated, causing the agarose to dissolve. 3. The resulting solution is then passed through a spin column by centrifugation. As this solution contains chaotropic salts or alcoholic solvents, the DNA will bind to the silica membrane in the spin column. 4. The DNA on the spin column can then be washed using an ethanol solution and eluted into a collection tube using water or buffer. Watch a video on how to extract DNA from agarose gels. (2:43). Monarch DNA Gel Extraction Kit Protocol – View on Youtube Use what you have learned about DNA isolation techniques discussed in this section to answer the question. Why are lysis and neutralization steps not needed for gel extraction? Dr. Lohans’ Response: In a gel extraction-based DNA purification, the DNA has already been isolated from a bacterial cell, and so it is not necessary to lyse open a bacterial cell to extract DNA. As lysis is not required, there is no need to neutralize the mixture. While the neutralization solution contains chaotropic salts which promote the binding of DNA to the silica membrane in a spin column, the buffer used to dissolve agarose also contains chaotropic salts and organic solvents, and performs the same function. In this section you learned about four methods used to purify DNA samples: phenol-chloroform extraction, ethanol precipitation, solid phase extraction, and gel extraction. In the next section, you will explore how these DNA samples can be analysed. Page Links: https://youtu.be/7Xgy5_i6iOc https://youtu.be/eAiansbvGVU ANTIBIOTIC RESISTANCE LAB|MICR 290 M03 PAGE 27 MODULE 03 COMPANION GUIDE MICR 290 End of Section 02 ANTIBIOTIC RESISTANCE LAB|MICR 290 M03 PAGE 28 MODULE 03 COMPANION GUIDE MICR 290 SECTION 03: ANALYSIS OF D NA After preparing a sample of DNA, whether by purification or other methods, it is important to know the concentration, the purity, and the identity of the DNA before moving forward with other experiments. DNA samples can be analyzed by U V-Vis spectrophotometry and agarose gel electrophoresis, as you will learn in this section. U V-Vis Absorbance In Module 01, you learned about the use of spectrophotometry for measuring the growth of bacterial cultures (i.e. turbidimetry). Remember that spectrophotometers shine a beam of light of a certain wavelength through a liquid sample, and measure how much light is absorbed. Spectrophotometers measure the absorbance of light by a sample solution.1 U V-Vis Spectrophotometry - Concentration Spectrophotometers can be used to measure the amount of DNA in a sample. While turbidimetry and Bradford assays involve the measurement of visible light (typically with a wavelength of around 600 n m), DNA can be quantified based on the absorbance of ultraviolet (U V) light. DNA has a maximum absorbance at a wavelength of 260 n m. By passing 260 n m light through a sample and measuring the extent to which this light is absorbed, the concentration of DNA can be determined. A U V-Vis spectrum for DNA with maximum absorbance at 260 n m. ANTIBIOTIC RESISTANCE LAB|MICR 290 M03 PAGE 29 MODULE 03 COMPANION GUIDE MICR 290 These spectrophotometric measurements are based on the Beer-Lambert law: A=εlc Where: A is the absorbance measured by the spectrophotometer. ε (epsilon) is the extinction coefficient, which relates to how well the molecule that is being quantified is able to absorb light. l is the path length of the sample (i.e. the distance that the beam of light traverses through the sample). c is the concentration of the molecule that absorbs lights. Based on this equation, absorbance and concentration are proportional. So, for example, if the concentration is doubled, the absorbance is also doubled, as shown in the graph. There are limits to how well the Beer-Lambert law works, and this linear relationship between concentration and absorbance does not apply at all concentrations. The concentrations of small molecules in solutions are often measured in terms of their molarity, or the number of moles per liter. However, the concentration of DNA is typically represented in terms of n g/μ L (nanograms per microliter). Limitations: Spectrophotometers measure the total amount of DNA in a sample, but are unable to directly measure the number of DNA molecules in the sample. A sample that contains a small number of long DNA strands could absorb the same amount of U V light as a sample with a large number of short DNA strands, or a complex mixture of DNA strands of different sizes. Also, while U V-Vis absorbance can be used to measure the concentration of DNA in a sample (at least in terms of mass per volume), it cannot tell anything about the identity (i.e. the sequence and length) of these DNA molecules. The extinction coefficient (ε) relates the absorbance of a sample to the absolute number of nucleotides in the sample, and not to the length of the DNA strands that contain those nucleotides. ANTIBIOTIC RESISTANCE LAB|MICR 290 M03 PAGE 30 MODULE 03 COMPANION GUIDE MICR 290 U V-Vis Spectrophotometry - Purity U V-Vis spectrophotometry can also be used to look at the purity of DNA in a sample. While DNA has an absorbance maximum at 260 n m, proteins absorb U V light with an absorbance maximum at 280 n m. After measuring the U V-Vis spectrum of a sample, the absorbances at 260 n m and 280 n m can be compared in order to estimate the amount of DNA and protein present. The ratio of these absorbances, the 260/280 ratio, provides a measure of the purity of the DNA. A 260/280 ratio of 1.8 suggests that a DNA sample contains only minimal protein contaminants. While the absorbance maximum for DNA is at 260 n m, it also absorbs some U V light at 280 n m. When the 260/280 ratio is 1.8, the absorbance at 280 n m results mainly from the DNA molecules in the sample. Graph of optical density as a function of wavelength, highlighting the differences between the absorption spectra for DNA and proteins. In addition to proteins, other molecules in a sample can absorb U V light, including commonly used buffers. To account for these molecules in the absorbance measurements, a spectrophotometer can be blanked or zeroed using a sample of the buffer that does not contain any DNA. Then, when the DNA sample is measured, the absorbance that results from the buffer is subtracted from the total measurement. U V-Vis Spectrophotometry - Volume Conventional spectrophotometers require fairly large sample volumes for accurate measurements. Depending on the type of cuvette (the container containing the sample that is placed in the spectrophotometer), the volume required may be up to 1 mL. However, the volumes of DNA solutions are generally far smaller than this. One option is to prepare a dilution of the DNA sample, measure the absorbance of the dilution, and extrapolate the concentration of the initial sample. This has largely been overcome by the development of microvolume spectrophotometers (e.g. NanoDrops), which are able to measure the U V/Vis absorbance of 1 μL samples. Alternatively, fluorescence-based assays can be used to quantify small amounts of DNA. ANTIBIOTIC RESISTANCE LAB|MICR 290 M03 PAGE 31 MODULE 03 COMPANION GUIDE MICR 290 Adding a sample to a NanoDrop spectrophotometer. Agarose Gel Electrophoresis Electrophoresis is a technique that separates molecules according to their charge. In MICR 290, you will explore agarose gel electrophoresis (AGE), which is used to separate nucleic acids, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (S D S-PAGE), which is used to separate proteins. This module will focus on AGE, and S D S-PAGE will be covered in Module 06. AGE is used to separate DNA molecules according to their size by loading them into a solid matrix and applying an electric field. This solid matrix is made of agarose, which forms a gel-like material. Switch between a diagram and a photograph of a gel electrophoresis chamber. Illustration of an agarose gel electrophoresis chamber.2 Photograph of an agarose gel electrophoresis chamber. ANTIBIOTIC RESISTANCE LAB|MICR 290 M03 PAGE 32 MODULE 03 COMPANION GUIDE MICR 290 Learn how an agarose gel tray is prepared for electrophoresis. Step 1 – Agarose Gel Preparation Illustration of how to set up an agarose gel tray.3 An agarose gel is prepared by heating solid agarose powder in a buffer, causing the agarose to dissolve. The agarose solution is then poured into a casting tray which defines the shape of the gel. As the solution cools, it solidifies, forming a block of semi-porous agarose gel. Step 2 – Agarose Gel Casting Illustration of how wells are formed using a comb in an agarose gel tray. 3 In order to load a sample of DNA into an agarose gel, wells are made in the surface of the gel. This is done using a comb, which makes a series of regular and defined holes in the gel surface. The comb is fitted onto the casting tray when the molten agarose solution is poured into the tray. Then, as the agarose solution cools, it solidifies around the “teeth” of the comb, leaving wells in the surface of the gel. Step 3 – Sample Loading Illustration of how samples are added to wells in an agarose gel tray. 3 ANTIBIOTIC RESISTANCE LAB|MICR 290 M03 PAGE 33 MODULE 03 COMPANION GUIDE MICR 290 Samples containing DNA are pipetted directly into the wells. An agarose gel can contain many wells, and the number and size of the wells is determined by the comb used. Each well has its own lane, representing the pathway that DNA travels between the well and the bottom of the gel. Different samples are loaded into different wells, and will run side-by-side in the lanes associated with these wells. The agarose gel electrophoresis chamber must be filled with a running buffer. The ions in the running buffer carry the electrical current that is required for electrophoresis to occur. The most common running buffer is known as TAE, which contains Tris, acetate, and ethylenediaminetetraacetic acid (EDTA). The components of the buffer help control the pH of the sample, protecting the DNA. In addition, the EDTA binds strongly to certain metal ions, deactivating many of the enzymes that can degrade DNA. Typically, the running buffer is used to dissolve the agarose when the gel is being prepared. DNA Migration When an electric field is applied, electrostatic forces pull DNA towards the positively charged part of the field (towards the anode). Recall that the phosphate backbone of DNA is negatively charged. DNA must travel through a complex network of pores in the agarose gel in order to reach the anode. Think about how the size of a DNA molecule might impact how easily it travels through agarose as you answer the following question. A sample containing DNA strands with three different lengths is loaded into the well of an agarose gel and an electric field is applied. The DNA strands are 263 b p, 537 b p, and 3472 b p in size. Predict the order of the bands that appear on the resulting agarose gel. a) Longer DNA will move the furthest distance so the correct order is: closest to the cathode – 3472 b p, 537 b p, 263 b p – closest to the anode. b) Longer DNA will move the furthest distance so the correct order is: closest to the cathode – 263 b p, 537 b p, 3472 b p – closest to the anode. c) Smaller DNA will move the furthest distance so the correct order is: closest to the cathode – 263 b p, 537 b p, 3472 b p – closest to the anode. d) Smaller DNA will move the furthest distance so the correct order is: closest to the cathode – 3472 b p, 537 b p, 263 b p – closest to the anode. e) AGE does not separate DNA by length and the order cannot be determined using the information provided. Feedback: d). Smaller DNA strands migrate through the gel matrix relatively quickly and easily, as they are less likely to be impeded by the material of the gel. On the other hand, longer DNA strands migrate more slowly, as they are more likely to be slowed down by the gel matrix. Therefore, agarose gel electrophoresis separates DNA molecules according to their size into discrete lines called bands. ANTIBIOTIC RESISTANCE LAB|MICR 290 M03 PAGE 34 MODULE 03 COMPANION GUIDE MICR 290 Illustration of an agarose gel electrophoresis chamber, showing the separation of DNA molecules according to their size.2 The amount of agarose in a gel determines the size of the pores in the gel. Most gels contain 1% agarose, meaning that they contain 1 g of agarose per 100 mL of buffer. While 1% agarose gels are good at separating relatively large pieces of DNA, they do not work as well for smaller ones. Therefore, higher percentage agarose gels (e.g. 2%) are often used to separate small DNA molecules. These gels have smaller pores, and are better able to resolve smaller pieces of DNA. Illustration of a completed agarose gel electrophoresis experiment, highlighting the role of a DNA ladder.3 Although AGE separates DNA molecules according to their size, this alone cannot be used to determine the size of these molecules. Therefore, DNA samples are typically run alongside a DNA ladder, which is a mixture of DNA molecules of known size (e.g. 500 b p, 1 k b, 2 k b, 3 k b, and so on). If an unknown DN A sample is run on an agarose gel alongside a DNA ladder, the size of the DNA molecules in the unknown sample can be determined by comparing their locations to the bands in the ladder. DNA Visualization If a DNA sample is separated using an agarose gel, how are the experimental results visualized? To monitor DNA migration, DNA stains (such as ethidium bromide or SYBR Safe) are added to the agarose gel. These stains are molecules that intercalate into DNA, inserting into the hydrophobic core of double-stranded DNA. When a molecule of stain is in this hydrophobic environment, it becomes fluorescent (i.e. it is excited by light of one wavelength, and emits light of a different wavelength). ANTIBIOTIC RESISTANCE LAB|MICR 290 M03 PAGE 35 MODULE 03 COMPANION GUIDE MICR 290 Although the agarose gel is full of DNA stain, only those parts that contain DNA are fluorescent, and can be visualized using a gel imager. The fluorescent parts of an agarose gel that contain DNA are the bands. Because these DNA stains intercalate into DNA, there is a possibility that they will interfere with DNA replication and lead to mutations in living organisms. Therefore, special precautions are made when working with these stains to minimize the risk of accidental exposure. This photograph shows a completed electrophoresis run on an agarose gel. The gel was stained with ethidium bromide and photographed under ultraviolet light. Unknown samples were added to the gel. In addition, DNA ladders were added on the two outside lanes to provide adequate reference sizes. 3 AGE can also be used to measure the purity of a DNA sample, revealing the number and size of the DNA molecules present. However, the resolution of AGE is somewhat limited. If two different DNA strands of slightly different sizes are analyzed by AGE, it is often not possible to distinguish between them. The fluorescence intensity of a DNA band in a stained agarose gel is proportional to the amount of DNA present in that band. Therefore, AGE can also be used to quantify the DNA in a sample, using an approach known as densitometry. This technique compares the intensity of an unknown sample to the intensity of a standard that contains a known amount of DNA (e.g. the bands in a DNA ladder). Software is used to establish a relationship between the band intensity and the amount of DNA for the standard sample, thereby allowing the amount of DNA in an unknown sample to be related to its intensity. DNA Fingerprinting The size and nucleotide sequence of the DNA in a bacterial cell is characteristic of the type of bacteria, with different species of bacteria having chromosomes and plasmids of different sizes and sequences. In principle, the size of the chromosome and plasmids could be used to identify a bacteria. However, agarose gel electrophoresis cannot resolve DNA molecules as large as a bacterial chromosome. ANTIBIOTIC RESISTANCE LAB|MICR 290 M03 PAGE 36 MODULE 03 COMPANION GUIDE MICR 290 Agarose gels are usually used to separate DNA molecules ranging from 100 bp to 10 k b in size, whereas a bacterial chromosome is typically several M b in size. To overcome this, enzymes known as restriction enzymes (or restriction endonucleases) can be used. Restriction enzymes (which are covered in Module 05) recognize and cut DNA at particular nucleotide sequences. For example, the restriction enzyme EcoRI recognizes the DNA sequence GAATTC, cutting between G and A. By treating a bacterial chromosome with restriction enzymes, it can be digested into pieces that are small enough to be separated on an agarose gel. The restriction enzyme EcoRI cuts the recognized DNA sequence.4 The distribution of restriction enzyme recognition sites (which are typically six base pairs long) in chromosomal DNA varies between different kinds of bacteria. Digesting a bacterial chromosome with a restriction enzyme and running the fragments on an agarose gel will result in a distinct pattern of DNA bands. Digestion of the chromosome from a different bacterium with the same restriction enzyme may give rise to a different pattern of DNA bands. Thus, the pattern of bands is a DNA fingerprint characteristic to that kind of bacteria. This can be applied to identify an unknown bacterial isolate. The DNA of this unknown isolate is digested with a restriction enzyme, and analyzed by agarose gel electrophoresis. By comparing this DNA fingerprint to those from known bacterial species, it may be possible to identify the unknown isolate. Southern Blotting A major drawback of agarose gel electrophoresis is that it provides information regarding the sizes of D NA molecules, but nothing about the nucleotide sequences of these molecules. Additionally, overlapping bands complicate the analysis of AGE. For example, a mixture of two different DNA strands of the same size will only give a single band on an agarose gel. These drawbacks can be addressed using a technique known as a Southern blot, which provides information regarding the sequence of the DNA molecules in an agarose gel. This technique makes use of a probe, consisting of a short DNA sequence attached to a label (often a radioactive tag or a fluorescent molecule). The nucleotide sequence of this probe is designed to complement whatever sequence is of interest (e.g. if the goal is to detect the presence of a particular gene in a sample, a DNA probe will be chosen that complements this gene). By using a probe that only binds to a particular DNA ANTIBIOTIC RESISTANCE LAB|MICR 290 M03 PAGE 37 MODULE 03 COMPANION GUIDE MICR 290 sequence, only those bands on the gel that contain this sequence will be labeled with the probe, allowing for their detection using the tag attached to the probe (e.g. through fluorescence). A detailed discussion on Southern blotting is beyond this course, although the protocol for a related technique (Western blotting) will be covered in Module 06. Go through the steps to learn the general protocol for Southern blotting. Step 1 – Separation of DNA Fragments by AGE A sample of DNA is first separated by agarose gel electrophoresis. Notice that a DNA ladder is included for reference.3 Step 2 – Transfer of DNA to a Membrane Then, the DNA bands in the agarose gel are transferred from the agarose gel to a membrane (typically made of nitrocellulose or nylon). The gel is sandwiched between a membrane and a reservoir of buffer. The buffer moves up from the reservoir and through the gel, carrying the DNA fragments from the gel to the membrane. The DNA molecules are then cross-linked to the membrane surface by using U V light.3 Step 3 – Membrane Exposed to Probe ANTIBIOTIC RESISTANCE LAB|MICR 290 M03 PAGE 38 MODULE 03 COMPANION GUIDE MICR 290 The membrane is washed in a solution containing a probe, a short piece of DNA complementary to the sequence of interest. The probe is labeled or tagged with a fluorescent dye or a radioactive label so that the location of DNA fragments to which it hybridizes can be visualized. The bands of the membrane that “light up” contain a DNA sequence that is complementary to the probe.3 Match each question to the most appropriate experimental technique. Choices: Southern Blot U V Spectrophotometry AGE DNA Fingerprinting Does this sample contain DNA that contains gene X? How long are each of the DNA strands in this sample? What type of bacteria formed this colony? What is the concentration of DNA in this sample? Feedback: Does this sample contain DNA that contains Southern Blot gene X? How long are each of the DNA strands in this AGE sample? What is the concentration of DNA in this sample? U V Spectrophotometry What type of bacteria formed this colony? DNA Fingerprinting This section explored experimental methods for analyzing DNA. You learned about U V spectrophotometry, agarose gel electrophoresis, DNA fingerprinting, and Southern blotting. In the next section, you will examine DNA sequencing data. References: 1. Chemistry LibreTexts. (2013, October 2). Spectrophotometry. Retrieved August 29, 2020, from https://chem.libretexts.org/Bookshelves/Physical_and_Theoretical_Chemistry_T ANTIBIOTIC RESISTANCE LAB|MICR 290 M03 PAGE 39 MODULE 03 COMPANION GUIDE MICR 290 extbook_Maps/Supplemental_Modules_(Physical_and_Theoretical_Chemistry)/Kinetic s/Reaction_Rates/Experimental_Determination_of_Kinetcs/Spectrophotometry 2. Drabik, A., Bodzoń-Kułakowska, A., & Silberring, J. (2016). 7 - Gel Electrophoresis. In Proteomic Profiling and Analytical Chemistry (Second Edition), 115–143. Elsevier. Retrieved August 29, 2020, from https://doi.org/10.1016/B978-0-444-63688- 1.00007-0 3. Keenleyside, W. (2019). Chapter 13.2: Microbiology: Canadian Edition. Pressbooks. Retrieved August 29, 2020, from https://ecampusontario.pressbooks.pub/microbio/chapter/visualizing-and- characterizing-D N A-rna-and-protein/ 4. Mr. Lee’s Biology 12 Site. (2018). Restriction enzyme EcoRI. Retrieved August 29, 2020, from https://mrleehamberbio12.wordpress.com/restriction-enzyme-ecori/ End of Section 03 ANTIBIOTIC RESISTANCE LAB|MICR 290 M03 PAGE 40 MODULE 03 COMPANION GUIDE MICR 290 SECTION 04: D NA SEQUENCING DATA In Module 02, Sanger sequencing and next-generation sequencing methods were briefly introduced. This section will focus on the generation and analysis of sequencing data from Sanger sequencing and next-generation sequencing. Sanger Sequencing Sanger sequencing can be used to determine DNA sequences that are around 500-1000 base pairs (b p) in length. Considering that bacterial chromosomes are typically several megabase pairs (M b) in size, the region covered by Sanger sequencing is quite small. In fact, considering that one amino acid is encoded by three base pairs (i.e. one codon), one Sanger sequencing run cannot usually cover the entire sequence of a gene that encodes a protein larger than ~300 amino acids. To compensate for this, several Sanger sequencing reactions can be carried out in parallel, with each reaction covering a different section of DNA. The region of DNA to be sequenced is specifically controlled by designing and using an oligonucleotide primer, a short single-stranded DNA molecule that is complementary to the DNA of interest. There are hundreds of different software tools used to analyze DNA sequences, providing information about the genes that they encode, their similarity to sequences in online databases, and much more. However, for these tools to work properly, DNA sequences often must be formatted correctly. The most common sequence format for DNA sequences (and protein sequences) is FASTA format. This is simply a text file in which each sequence is given a title using a greater-than symbol (>). Here is a simple DNA sequence in FASTA format: > Sequence 1 ATGCTGACGTACGTGCATGGTACAT Using FASTA formatting, you can include multiple sequences in a single file, as long as they are properly identified using a > symbol. For example: > Sequence 1 ATGCTGACGTACGTGCATGGTACAT > Sequence 2 ATGGTCGACCTGATGCGTGATCATC Sanger sequencing data is commonly provided in FASTA format. Next-Generation Sequencing While Sanger sequencing provides only a short DNA sequence determined by the oligonucleotide primer used, next-generation sequencing methods reveal the sequences of all of the DNA molecules in a sample. To accomplish this, next-generation methods actually determine millions to billions of short ANTIBIOTIC RESISTANCE LAB|MICR 290 M03 PAGE 41 MODULE 03 COMPANION GUIDE MICR 290 DNA sequences (or reads), ranging from ~20 to 400 b p in length according to the particular technology. The next step is to combine these short DNA reads into a complete DNA sequence. This process, known as assembly, is computationally demanding, requiring the analysis of millions or billions of reads. Next-generation sequencing reads are random sequences of DNA, and so many of the reads have overlapping sequences. The software used for sequence assembly will look for these overlapping sequences, piecing them together to assemble the complete DNA sequence. Assembly 1 Consider the sequencing of a short 100 b p strand of DNA. One sequencing read may cover nucleotides 1-50 in this 100 b p sequence, a second read may cover nucleotides 25-75, and a third read may cover nucleotides 50-100. The DNA assembly software will look at the overlap of the first read with the second read, and assemble these two reads into a single sequence spanning nucleotides 1-75. Schematic representation of how the first two of three DNA sequence reads would be assembled into an intermediate fragment. Assembly 2 Next, the software will compare the third read (nucleotides 50-100) with the combined first and second reads (nucleotides 1-75), and based on the overlap of nucleotides 50-75, will assemble them to provide the full sequence from 1-100. Schematic representation of how a previously assembled fragment of DNA is further assembled with a third read into the full-length DNA fragment. ANTIBIOTIC RESISTANCE LAB|MICR 290 M03 PAGE 42 MODULE 03 COMPANION GUIDE MICR 290 A similar process occurs on a much larger scale for actual next-generation sequencing. The amount of overlapping reads (or the coverage) provides a measure of the quality of the assembled DNA sequence. Schematic representation of how computer software assembles DNA reads, showing how regions of high or low quality can be achieved. Answer the question using what you have learned about next-generation sequencing and the example used previously. What if the second read is missing, and only the first read (nucleotides 1-50) and the third read (nucleotides 50-100) are present? Dr. Lohans’ Response: In this case, the sequence assembler is unable to determine that nucleotides 1- 50 and nucleotides 50-100 are adjacent in the complete DNA sequence; there is no read that bridges these two nucleotide sequences. If the assembly software is unable to combine all of the reads into a single DNA sequence, it will generate contigs, DNA sequences which cannot be assembled with other sequences. In this example, contig 1 would be nucleotides 1-50, and contig 2 would be nucleotides 50-100. Schematic representation of the generation of contigs from example DNA sequences that are unable to be assembled together due to gaps in DNA reads. Next-generation sequencing often results in many different contigs. This may result from the presence of different DNA molecules in the sample, or if the sequencing data is unable to connect different parts of the same sequence. When sequencing a DNA sample from bacteria, the plasmids and chromosomal DNA will appear as separate contigs. Similar to Sanger sequencing, next-generation sequencing results are typically provided in FASTA format, with each contig separated with a > symbol and a sequence title. ANTIBIOTIC RESISTANCE LAB|MICR 290 M03 PAGE 43 MODULE 03 COMPANION GUIDE MICR 290 This section introduced the generation and analysis of sequencing data for both Sanger sequencing and next-generation sequencing. In the final section, you will learn about two online bioinformatics tools used to further analyze these sequences. End of Section 04 ANTIBIOTIC RESISTANCE LAB|MICR 290 M03 PAGE 44 MODULE 03 COMPANION GUIDE MICR 290 SECTION 05: BIOINFORMATICS APPROACHES FOR D NA SEQUENCE ANALYSIS Bioinformatics is a scientific field centered on the use of computational approaches to investigate and analyze biological systems. There are hundreds of different tools for analyzing D NA sequences (and protein sequences), available as part of commercial software packages, or for free online. This module will cover two common tools: ExPASy Translate and BLAST. ExPASy Translate Tool Sequencing data is often used to characterize the proteins encoded by a particular DNA sequence. However, how is it possible to determine which parts of a DNA sequence encode for protein? Open ExPASy. Keep it open in a separate tab in your browser so you can use it to assist your learning throughout this section. The ExPASy Translate tool is useful for identifying genes (also known as open reading frames) in a sequence of DNA – ExPASy Translate The input for the Translate tool is the DNA sequence in FASTA format. As an example, try translating the nucleotide sequence in Translation Example 1 using ExPASy Translate. Copy the sequence into the text box titled “DNA or RNA sequence”. Then use the “TRANSLATE!” button. > Translation Example 1 ATGGAATTGCCCAATATTATGCACCCGGTCGCGAAGCTGAGCACCGCATTAGCCGCTGCATTGATGCTGAGCGG GTGCATGCCCGGTGAAATCCGCCCGACGATTGGCCAGCAAATGGAAACTGGCGACCAACGGTTTGGCGATCTGG TTTTCCGCCAGCTCGCACCGAATGTCTGGCAGCACACTTCCTATCTCGACATGCCGGGTTTCGGGGCAGTCGCTT CCAACGGTTTGATCGTCAGGGATGGCGGCCGCGTGCTGGTGGTCGATACCGCCTGGACCGATGACCAGACCGC CCAGATCCTCAACTGGATCAAGCAGGAGATCAACCTGCCGGTCGCGCTGGCGGTGGTGACTCACGCGCATCAGG ACAAGATGGGCGGTATGGACGCGCTGCATGCGGCGGGGATTGCGACTTATGCCAATGCGTTGTCGAACCAGCTT GCCCCGCAAGAGGGGATGGTTGCGGCGCAACACAGCCTGACTTTCGCCGCCAATGGCTGGGTCGAACCAGCAA CCGCGCCCAACTTTGGCCCGCTCAAGGTATTTTACCCCGGCCCCGGCCACACCAGTGACAATATCACCGTTGGGA TCGACGGCACCGACATCGCTTTTGGTGGCTGCCTGATCAAGGACAGCAAGGCCAAGTCGCTCGGCAATCTCGGT GATGCCGACACTGAGCACTACGCCGCGTCAGCGCGCGCGTTTGGTGCGGCGTTCCCCAAGGCCAGCATGATCGT GATGAGCCATTCCGCCCCCGATAGCCGCGCCGCAATCACTCATACGGCCCGCATGGCCGACAAGCTGCGCTGA The output from this example should be: ANTIBIOTIC RESISTANCE LAB|MICR 290 M03 PAGE 45 MODULE 03 COMPANION GUIDE MICR 290 Translation Example 1 output. As you will see in Module 04, the directionality of a DNA sequence is very important. This directionality is related to the chemical backbone of DNA. As you have learned, a strand of DNA has two ends: a 5ʹ end and a 3ʹ end. When a DNA sequence is provided, it almost always begins with the 5ʹ end and finishes with the 3ʹ end. Genes begin with a start codon and end with a stop codon. The translate software identifies potential start codons (A T G, which translates to M) and stop codons (TAG, TAA, T G A, represented by a dash -) in the inputted DNA sequence. If a start codon and a stop codon are in frame, ExPASy Translate will highlight the sequence of amino acids between them in pink. Recall that 'in frame' means they are separated by a number of base pairs that is a multiple of three, with three nucleotides making up one codon. Reflect on the question before viewing Dr. Lohans’ response. Why does the Translate software output six different sequences, labeled 5ʹ3ʹ Frames 1, 2, and 3, and 3ʹ5ʹ Frames 1, 2, and 3? Dr. Lohans’ Response: These represent the six different possible reading frames for a DNA sequence. This is because one amino acid is encoded by a codon made up of three nucleotides, and because DNA is antiparallel. Using the output provided by ExPASy Translate for the Translation Example 1 sequence, answer the question. Which reading frame is most likely to contain a gene? a) 5’ 3’ Frame 1 b) 5’ 3’ Frame 2 c) 5’ 3’ Frame 3 Feedback: ANTIBIOTIC RESISTANCE LAB|MICR 290 M03 PAGE 46 MODULE 03 COMPANION GUIDE MICR 290 a). The most likely reading frame to contain a gene is Frame 1. This is because it has the longest open reading frame (highlighted in pink). Frame 2 and Frame 3 have some open reading frames, but they are very short. This is because they have more stop codons than Frame 1. How do the different reading frames correspond to different amino acid sequences? Try entering the Translation Example 2 DNA sequence into the ExPASy Translate tool. Use the “reset” button. Copy the sequence into the text box titled “DNA or RNA sequence” then use the “TRANSLATE!” button. > Translation Example 2 AATGCGTAGCTGACGTAGTTGAC As you can see from the output provided by ExPASy Translate for the Translation Example 2 sequence, each frame corresponds to a different amino acid sequence. 5ʹ3ʹ Frame 1 interprets this sequence starting at the very beginning, with codons AAT, G C G, TAG, C T G, and so on. 5ʹ3ʹ Frame 2 interprets this sequence beginning at the second nucleotide, with codons A T G, C G T, A G C, etc. 5ʹ3ʹ Frame 3 begins at the third nucleotide, with codons T G C, G T A, G C T, etc. The amino acid sequences for the 3’5’ frames are provided because DNA is double-stranded and some genes are present on one strand, while others are present on the other strand. Here is the DNA sequence in Translation Example 2 with its complementary strand. Note the antiparallel orientation of the two strands, with the top strand reading out from 5ʹ to 3ʹ (left to right), and the bottom strand reading out from 3ʹ to 5ʹ. 5’ AATGCGTAGCTGACGTAGTTGAC 3’ 3’ TTACGCATCGACTGCATCAACTG 5’ The codons in DNA are read from 5ʹ to 3ʹ. So, to see if the bottom strand contains any genes, the sequence must be reversed such that it is oriented 5ʹ to 3ʹ: 5’ GTCAACTACGTCAGCTACGCATT 3’ The Translate tool analyzes this reversed sequence to generate the reading frames labeled as 3ʹ5ʹ Frames 1, 2, and 3. The software is looking for genes that are encoded for by the DNA sequence that is the reverse complement to the DNA sequence provided as input. This sequence is first reversed (e.g. from A T G to G T A), and the complement of the reverse sequence is determined (e.g. from G T A to CAT; C is the complement of G, A is the complement of T, and so on). In Translation Example 2, 3ʹ5ʹ Frame 1 starts with the codon G T C, 3ʹ5ʹ Frame 2 skips the first G and starts with the codon T C A, and 3ʹ5ʹ Frame 3 skips G T and starts with the codon CAA. BLAST Basic Local Alignment Search Tool (BLAST) is a bioinformatic tool that measures the level of similarities between different sequences of nucleotides or amino acids. The user enters a query sequence, which the BLAST tool compares against a database of known sequences. BLAST then ANTIBIOTIC RESISTANCE LAB|MICR 290 M03 PAGE 47 MODULE 03 COMPANION GUIDE MICR 290 outputs a list of the sequences from the database that most closely resemble the input sequence. This is invaluable for identifying nucleotide sequences and proteins. Open BLAST. Keep it open in a separate tab in your browser so you can use it to assist your learning throughout this section. You can access BLAST on the National Center for Biotechnology Information (N C B I) website. – Blast For analyzing nucleotide sequences, BLASTn (nucleotide BLAST) is used. BLASTn performs a sequence alignment, aligning the input sequence with sequences in the database, and scoring how well they match. In the last module, you learned how 16SrRNA is used to identify bacterial species, and how DNA sequencing methods can be used to determine this sequence. BLAST represents the next step in this analysis, allowing the sequencing results to be compared against a database of 16SrRNA sequences covering a broad range of different bacterial species. As an example, suppose that you obtained the 16SrRNA sequence titled Example A using Sanger sequencing. This represents only a small portion of a complete 16SrRNA sequence, due to the limitations of Sanger sequencing. > Example A TTATCGGAGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGGACAGA TGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATA ACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAAACATAAAAGGTGGCTTCGGCT ACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAA Try analyzing this sequence using BLASTn. Copy the sequence into the text box titled “Enter accession number(s), gi(s), or FASTA sequence(s)” then use the “BLAST” button at the bottom of the webpage. BLASTing this sequence brings up a long list of hits, all of which are annotated as belonging to different strains of Bacillus subtilis. Therefore, by determining the 16SrRNA sequence of an unknown isolate, the species of the isolate can be easily determined using BLAST. The hits at the top of this list are those that most closely match the query sequence, with the level of similarity decreasing going down the list. This is represented quantitatively using the Expect value (or E value). The lower the E value, the higher the level of similarity. In this particular example, all of the E values are extremely small (7e-143, or 7 x 10-143), and so all of these hits very closely resemble the query sequence. Use the top hit to explore BLASTn further. You should now see a screen like this: ANTIBIOTIC RESISTANCE LAB|MICR 290 M03 PAGE 48 MODULE 03 COMPANION GUIDE MICR 290 Example A BLASTn output for Bacillus subtilis JCL16. BLASTn has performed a sequence alignment, aligning the input sequence (Query) with a nucleotide sequence found in the chromosome of Bacillus subtilis strain J C L16 (Sbjct). Note the numbers at the start of the Query and Sbjct lines: these represent the numbers assigned to the nucleotides of the two sequences. The input started with TTATCGG, and so these are assigned as nucleotides 1 - 7 in the Query. In the B. subtilis chromosome, this DNA sequence begins at nucleotide 9487. The vertical lines between the nucleotides in the Query sequence and the Sbjct sequence indicate positions that are an exact match. If there was a mismatch (e.g. A in the Query, T in the Sbjct), a vertical line would not be displayed between them. Note at the top of this image the Identities and Gaps descriptors. The identities of all 280 nucleotides in the query sequence exactly match all 280 nucleotides in the database sequence, and so the percent sequence identity is 100%. The Gaps descriptor will be explored more in the next example. Now that you have explored nucleotide BLAST return to the BLAST homepage and select protein BLAST. The BLASTp (protein BLAST) tool can be used to compare an amino acid sequence against a database of proteins. For example, BLASTp can be used to study beta-lactamases, enzymes that degrade beta- lactam antibiotics and confer bacteria with antibiotic resistance. There are thousands of different beta- lactamase variants, all of which have different amino acid sequences. Here is the amino acid sequence for the beta-lactamase OXA-405 in FASTA format: > OXA-405 MRVLALSAVFLVASIIGMPAVAKEWQENKSWNAHFTEHKSQGVVVLWNENKQQGFTNNLKRANQAFLPASTFKIPN SLIALDLGVVKDEHQVFKWDGQTRDIATWNRDHNLITAMKYSVVPVYQEFARQIGEARMSKMLHAFDYGNEDISGN ANTIBIOTIC RESISTANCE LAB|MICR 290 M03 PAGE 49 MODULE 03 COMPANION GUIDE MICR 290 VDSFWLDGGIRISATEQISFLRKLYHNKLHVSERSQRIVKQAMLTEANGDYIIRAKTGYSPKIGWWVGWVELDDNVWF FAMNMDMPTSDGLGLRQAITKEVLKQEKIIP Input this sequence into BLASTp similar to how you did for Example A in BLASTn. However, before you press the BLAST button, click on “+ Algorithm parameters” (below the BLAST button) and change the “Max target sequences” drop-down menu to 500. Inputting this sequence into BLASTp brings up a long list of beta-lactamases, all of which are similar to OXA-405. The top entry on this list corresponds to the database entry for OXA-405, which is as expected. As you go down the list, the entries begin to become more and more dissimilar from the OXA-405 sequence. Take a closer look at the hit titled “ OXA-48 family carbapenem-hydrolyzing class D beta-lactamase OXA-436”, which is the beta-lactamase OXA-436. This hit is relatively low on the list, and so it is not one of the closest matches to OXA-405 in terms of amino acid sequence. In the “Descriptions” tab use the select all button off so none of the list items are checked, then scroll down the list to find “ OXA-48 family carbapenem-hydrolyzing class D beta-lactamase OXA-436 [Gammaproteobacteria]” and use the box beside it. Once you have selected the correct entry, go to the Alignments tab. Your screen should now look like this: OXA-405 BLASTp output. ANTIBIOTIC RESISTANCE LAB|MICR 290 M03 PAGE 50 MODULE 03 COMPANION GUIDE MICR 290 This window shows the sequence alignment of OXA-405 (Query) with OXA-436 (Sbjct). Both of these sequences begin with the amino acid methionine (M) at position 1. Although both sequences end with the same amino acid sequence (KIIP), you can see that the lengths of the sequences are different. Use the output from your BLAST search to answer the questions. 1 of 4: How long is the Query sequence in this example? (Please use numbers only in your response.) Feedback: 261. The length of the Query sequence (i.e. the input sequence) is given by the number at the end of the last line of amino acids that has the title “Query.” In this example, the Query is 261 amino acids in length, and the Sbject is 265 amino acids in length. You can see the gap in the sequence alignment between amino acids 181 and 236 in the Query sequence - this is represented by the four hyphens (-). This indicates that the Sbjct sequence has a stretch of four amino acids that do not correspond to amino acids present in the Query sequence. Gaps are represented numerically by the Gaps parameter. 2 of 4: What is the Gaps parameter in this example? (Use the format #/#. For example, 27/400) Feedback: 4/265. The Gaps value is given in the top right corner of the BLAST output. In this example, the Gaps value is 4/265, meaning that the longest sequence is 265 amino acids, and there is one or more gap in the sequence alignment for a total gap of 4 amino acids (represented by hyphens in the Query sequence). Many of the amino acids in OXA-405 (Query) and OXA-436 (Sbjct) are the same. For those amino acids that match exactly, the letter of that amino acid is placed between the two rows in the sequence alignment. For example, both sequences have a methionine (M) in position 1, and so an M is placed between the two sequences. However, looking through this sequence alignment, you can see that several positions have a blank space or a + symbol between the Query and Sbjct lines. This shows that the Query sequence and the Sbjct sequence have different amino acids at this position. The + symbol shows that the two amino acids have similar chemical properties, such as serine (S) and threonine (T). A blank space indicates that the two amino acids are not very similar, such as lysine (K) and threonine (T). Similar to the BLASTn output, BLASTp calculates Identities, which is the percent sequence identity. This number is obtained by subtracting all of the blank spaces, + symbols, and gaps from the total number of amino acids of the longer sequence (OXA-436, Sbjct), and dividing it by the total number of amino acids of the longer sequence. 3 of 4: What is the Identities value in this example? (Use the format #/#. For example, 27/400) Feedback: ANTIBIOTIC RESISTANCE LAB|MICR 290 M03 PAGE 51 MODULE 03 COMPANION GUIDE MICR 290 239/265. The Identities value is given above the sequence alignment in the BLAST output. In this example, the Identities value is 239/265, meaning that the two sequences have a percent sequence identity of 90%. The 26 different positions (265 - 239) are comprised of gaps (4), amino acids that are not similar (9), and amino acids with some similarity (13). BLASTp also calculates a Positives value. Unlike Identities, Positives does not subtract the amino acids indicated with + symbols from the total number of amino acids, only subtracting blank spaces and gaps. 4 of 4: What is the percent Positives value in this example? (Use the format #%. For example, 45%) Feedback: 95%. The percent Positives value in this example is given above the sequence alignment, between the Identities and Gaps values. In this example, the Positives value is 252/265 or 95%, meaning that 95% of the amino acids are either the same or similar between the two sequences. The 13 different positions (265 - 252) are comprised of gaps (4) and amino acids that are not similar (9). BLAST uses a complex algorithm to rank sequences of nucleotides or amino acids based on the number of gaps, the percent sequence identity, and the similarity of amino acids (for protein sequences). This algorithm identifies the sequences in the database which give rise to the fewest gaps and the highest % sequence identity compared to the query sequence. BLAST is an invaluable research tool, allowing new nucleotide sequences or proteins to be rapidly compared to databases that cover millions of known sequences. There are many different applications for BLAST, including the identification of unknown bacterial species (based on 16SrRNA) and the prediction of protein function based on similarity to known proteins. In this section you learned about ExPASy Translate and BLAST, two bioinformatics tools used for analyzing nucleotide and amino acid sequences. You also used these tools with example sequences to explore the different parameters used to compare nucleotide and amino acid sequences. This concludes the content component of Module 03: Purification and Analysis of Bacterial DNA. In this module, you learned about the major mechanisms by which bacteria take up DNA and how these mechanisms can lead to the dissemination of antibiotic resistance. You also learned about several common methods by which DNA can be purified and analyzed. Lastly, you learned about the generation of sequencing data and several bioinformatics tools used to compare and identify sequences of nucleotides and amino acids. In the next module, you will learn how DNA can be amplified in a process known as the polymerase chain reaction (P C R), and how this process forms the basis of several other techniques, such as Sanger sequencing. Page Links: https://web.expasy.org/translate/ ANTIBIOTIC RESISTANCE LAB|MICR 290 M03 PAGE 52 MODULE 03 COMPANION GUIDE MICR 290 https://blast.ncbi.nlm.nih.gov/Blast.cgi End of Section 05 End of Module 03 ANTIBIOTIC RESISTANCE LAB|MICR 290 M03 PAGE 53

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