Exam 4 Review

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Feb 20, 2024

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Exam 4 Review 25 points + 10 extra effort Instructions: Answer using this document. Fill in the blanks or provide short answers to the following questions. Highlight your answers using color . You may use this sheet during your exam for quick reference. Lecture 16 and 17: 1. Describe the basic steps of PCR amplification and how much product is produced in each. Why is the curve shaped as it is? PCR works in the same way as the bacterial growth curve. It starts slow with only a little bit of product, but it quickly has exponential growth and there is doubling of product in every cycle. The final phase is exhaustion, in which the number of copies levels out, meaning something has run out, usually polymerase, and there is virtually nothing produced. 2. Why is transformation so important to biotechnology? Transformation allows bacteria to acquire DNA from the environment. This can be utilized in labs for protein production, DNA sequencing, and other analyses, and enables other technology like genome modification to occur. 3. Describe the basic components of a recombinant plasmid. What does each do? Why do we need each to use plasmids in the laboratory? The basic components of the recombinant plasmid are the DNA of interest, the MCS, required to get the DNA into the plasmid, the ori, which is required to amplify the plasmid, the selection marker, to make sure the only DNA looked at has the plasmid, and the reporter that sits across the MCS, which allows looking for bacteria that has taken up the plasmid with the insert. Plasmids are used in the lab because of their ability to be engineered to express proteins of interest. 4. What can scientists do with plasmids? Why are they so useful? Why do they contain selection genes? Plasmids allow geneticists to store, express, and manipulate the DNA they need. There are many genes in plasmids that increase fitness due to their part in survival for bacteria, and they can be used in PCR. 5. How did restriction enzyme evolve? What is their original biological purpose? How do we use them in the laboratory to build recombinant plasmids? Restriction enzymes evolved to protect bacteria from viruses. They are endonucleases that are produced by most/all bacteria to cut specific DNA sequences. They are used in laboratory to get the desired PCR product, creating matching sticky ends that can be added to plasmids to build recombinant plasmids. 6. What is a sticky end? How is it related to plasmids? A sticky end is a cut end of DNA that can be ligated to others to make a new DNA molecule. They are used to ligate PCR product to plasmids. 7. What is a blue-white screen? What does it tell you? A blue light screen is a test placed into a MCS to tell if an insert is in the plasmid. If the MCS isn’t disrupting the reporter gene, blue product will be produced; if the MCD insert is disrupting the reporter, blue product will not be produced. 8. What is an MCS? Why do they often contain a reporter gene? What is a blue-white screen? How would you analyze one? An MCS is a multiple cloning site that often contains a reporter gene so that the genetic insert can be detected by the blue-light screen. A blue light screen is a test placed into a MCS to tell if an insert is in the plasmid. If the MCS isn’t disrupting the reporter gene, blue product will be produced; if the MCD insert is disrupting the reporter, blue product will not be produced. This way, you can tell if the desired outcome is achieved. 9. How do you put a sticky end on a PCR product? Both the plasmid and PCR product are digested with the same restriction enzymes to form matching sticky ends, then both pieces are ligated together to form a recombinant plasmid. PCR

products can be designed with restriction enzyme sites in the 5’ ends of the PCR primers to generate a PCR product that can have sticky ends. 10. How does traditional gene engineering work? What are the various parts of the plasmid? What do they do? Traditional engineering uses standard recombination plasmid, which randomly sticks to portions of the genome and only works in ES cells. Targeted traditional engineering has the same parts as the random plasmid, but it also has homology arms that match where in the genome it needs to go, so it can enter a specific part of the genome; targeted engineering is much less efficient than random but generally works the same way. 11. What is the difference between plasmids for traditional random insertion and traditional targeted mutation? Do they contain all the same components? What components are shared and what components are unique? The major difference between random & targeted are the homology arms. Otherwise, they both contain the inserts necessary for insertion. The homology arms are used to find the similar regions of DNA, so HR can occur in the targeted region and the insert can be placed in a very specific area. 12. What is CRISPR-Cas9? How did it evolve? CRISPR-Cas9 is a type of genome editing that is used as a research tool. It evolved to protect bacteria from viruses, and relies on recombinant plasmids. 13. What are the parts of the CRISPR-Cas9 complex and what do they do? How do we use it to generate genetically modified organisms? The parts of the complex are the Cas9 enzyme which cuts DNA and the guide RNA that tells the Cas9 where to cut. There are two methods: repair only and repair & insert. The Cas9 will cut the DNA in a way that triggers repair in the cell after it is told where to go, and the NHEJ will resect the base pairs back then repair by joining the gap, this occurs in repair only. In repair & insert, HR is used, especially within a zygote; it will stick extra DNA in to repair the cut genome rather than just connect the two portions of cut DNA. 14. What is the difference between CRISPR repair only and repair+insert? What kind of change do they trigger in the genome? CRISPR repair only mutates DNA in small deletions and rearrangements, while repair + insert does large and small insertions. 15. How does CRISPR-Cas compare to traditional targeting? Can they both do the same thing? CRISPR is amendable to every species, while traditional targeting can only be used on mice due to the cells it targets. While traditional targeting can do large or small insertions, CRISPR can do those as well as small deletions and rearrangements. CRISPR also has a much higher success rate than traditional targeting and can be used in ES cells, zygotes, and tissues; traditional targeting can only be done in an ES cell. 16. Why does zygote injection (as opposed to ES cell modification) make genetic engineering more efficient? In zygote injection, the very early cells are being engineered, so there is a higher rate of incorporation than in ES cells. ES cells are transfected, which has a low success rate, and then are injected into a blastocyst, which are hopefully taken up and made into germ cells. Each portion of the ES modification has a low success rate, versus the zygote injection is much more effective because the engineering will eventually effect every cell. 17. What makes CRISPR the more commonly used engineering tool? CRISPR is amenable to every species and has a much higher efficiency rate. Therefore, it is better cost-wise and easier to use regardless of what species it is being used on, making it more favorable and commonly used. 18. What are the difference between gene modification and gene editing? What tools can you use for modification vs editing? Gene modification is not possible in nature while gene editing, while unlikely, is. Gene modification is usually large chunks of foreign DNA inserted into an organism’s DNA, which can be at random or targeted, and requires a recombinant plasmid. Gene editing is on a smaller

scale, since it is more simple, and is usually a point mutation or a recombination; gene editing is also targeted. Lecture 16/17 extra effort: 1. In the first stages of viral infection, the CRISPR-Cas system uses chopped up viral DNA. What other system we discussed likely generates the chopped up viral DNA? The other system is restriction enzymes: endonucleases are used to chop up the DNA when infected with a virus, and this DNA is used to fight the viral infection when it is injected into other cells. 2. PCR primers only REQUIRE annealing on their 3’ end to allow polymerase to synthesize DNA. Why is that? PCR mimics DNA replication; when polymerase is replicating DNA, it uses an RNA primer and adds onto the 3’ end. DNA primers are used in PCR, and the 3’ end needs to anneal so the RNA primer can recognize it and add onto it to complete replication. 3. What ethical considerations should be made when engineering food production animals? Why? The primary ethical consideration should be its effect on the environment and local ecosystem. This is because engineering animals for food production can be disastrous on already fragile ecosystems; much of the deforestation in Brazil can be attributed to cattle farming, for example, and engineering angus cows to better suit the environment can worsen this. Many people can be displaced or die because of this, as well, and overall, it may seriously contribute to climate change. The environment must come before efficiency of food production. Lecture 18: 1. Phenotypes can be one of two types. What are these types, how many genes regulate them, and how do we know this? Phenotypes can appear as either discrete, which is monogenic, and continuous, which is polygenic. Discrete phenotypes are “either/or,” which means there are two or more distinct populations of the phenotype; these are qualitative. Continuous phenotypes have a range on phenotypes with not discrete populations within; these are quantitative. 2. What are the components of a frequency distribution graph? How do you make one? What shapes can a distribution be? What if your population is too large to count? The components of a frequency distribution graph are the phenotypic measure and the number of individuals with that given phenotypic measure. The x-axis is built from a sample of the population which is representative of the whole population, and the phenotype must be quantitative. The y- axis is built using the frequency of the measured phenotype in the sample. The distribution is typically on a bell curve. 3. What information can you determine about a trait from a frequency distribution? What kinds of tools require frequency distributions? Using frequency distribution, the nature of the phenotype can be determined and the population can be described using the average & the range. A quantitative trait loci analysis requires frequency distributions to determine how genes impact a phenotype. 4. Quantitative genetics studies continuous phenotypes. 5. How can a disease be a quantitative trait? Disease frequency analyzed across populations show that there is a bell curve of how people are susceptible to disease. Disease can be analyzed as functions of exposure, age, genetics, etc. to determine if someone is susceptible to it, making it quantitative. 6. What are the three statistical measures used to describe a populations phenotype distribution? How are they related (or not)? Mean, variance, and standard deviation are used to describe distribution. In standard distribution, the mean will be the highest point in the bell curve. Variance means the difference from the highest to lowest numbers in the population. These two are not related at all; two populations can have the same mean but different variations. The standard deviation is the difference from the mean; it is a statistical measure of variance. Most people reside within 2 standard deviations of the mean, which means that while 90% of people are within this, there are 5% of people outside

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of 2 sd on the higher end, and 5% of people outside of 2 sd on the lower end. This is also not related to the mean, for the same reason of variation. 7. How do we use statistical measures to tell differences within and between a population? What is the role of standard deviation in this process? Provide an example. In women ages to 30-40, where individuals appear on the bell curve of how likely they are to get cancer can help determine their usefulness in studies. Outside two sd’s from the mean on either side can be studied to compare the genetics on what will make a woman in that age range more or likely to get cancer. 8. What is a statistical correlation? How do we quantify it? What is r? What does r tell us about the strength and/or direction of a correlation? Statistical correlation compares multiple characteristics: for example, two phenotypes. The x-axis measures characteristic 1, the y-axis measures characteristic 2. R is the correlation coefficient, which explains how the two characteristics are related, if at all: a large r indicates a strong correlation, while the smaller/more close to zero r is, the weaker the correlation is; if r is positive, the two characteristics are directly correlated, if they’re negative, they are inversely correlated. 9. Why are correlations fundamental to quantitative genetics? Correlations are fundamental to quantitative genetics because quantitative genetics correlates genotype to phenotype across populations. 10. What can you learn from a QTL or GWAS study? A QTL/GWAS can tell how many genes impact a phenotype, how much each gene impacts a phenotype, which genes are involved in the phenotype, and how a specific allele of a given gene impacts the phenotype. 11. Do either identify specific genes? How or how not? These do not identify specific genes, instead, they define regions of the genome that contribute to phenotypic variance. 12. What are the differences and similarities between QTL analyses and GWAS studies? Describe a situation where you may want to use one or the other. A GWAS is a QTL analysis that doesn’t require a quantitative trait and can be performed without breeding. This means, it can be used in humans, which is when it would typically be used; however, GWAS require large sample sizes. QTL utilizes a model organism, requires breeding, and has a true continuous phenotype; GWAS can be done for any organism, does not require breeding, and does not need a continuous phenotype. Both identify regions of the genome associated with a trait and may identify associated variants. Lecture 18 Extra effort: Describe a non-gene genomic element that may be identified by QTL as having a high LOD score and how that element may impact the trait being studied. QTL is used to identify regions of the genomes that contain variance associated with the phenotypes. If something has a high LOD score but no protein-coding genes, this can mean that there is something else that is affecting the trait. These can be enhancers and repressors that are far from genes but can loop around and still act on genes and influence their expression. A high LOD score can mean that it is affecting the way a gene is expressed, rather than the gene itself. Lecture 19: 1. Evolution is change in heritable characteristics of biological populations over successive generations . 2. What is the difference between macro and microevolution? Which can we observe in the lab and why? Macroevolution is dramatic biological changes across species (or taxa); it is the appearance of newer species from older ones and occurs on a large scale. Meanwhile, microevolution is heritable change within a population, resulting from genetic variation within individuals and occurs on a short time scale. Microevolution can be studied in a lab because it is on a smaller scale and occurs within a shorter amount of time. Macroevolution must be inferred because its difficult to measure directly.

3. What is a phylogenetic tree? How do we build one? What do the branch points, lines, and end points mean? What evolutionary process leads to the outcome a phylogenetic tree describes? A phylogenetic tree describes the relationship between species and the identification of the common ancestor. Mutation and selection are both used in phylogenetic trees, but it is mostly neutral mutations that lead to the difference within species that phylogenetic trees describe. Trees can be built by comparing genetic variation across many species. In a tree, branch points are the last common ancestor, branch lines can indicate time or evolutionary distance, and end points describe species. 4. What is the classic definition of a species? Why is it wrong? The classic definition is that if two different species mate, their offspring (if there is any) is not fertile. However, there is Neanderthals DNA in humans; the only way this can occur is if there is mating between Neanderthals and humans. This means that this definition of species must be wrong. 5. How is genetic variation related to evolution? How do individuals accrue genetic variation? Populations? Genetic variation causes differences in fitness within a population, which leads to natural selection for better traits. This can cause evolution. Variation within an individual is caused by genetic mutations, which can be either positive, neutral or negative. Within a population, variation is caused by frequency in genetic variants. This leads to gene flow or genetic drift, and can drive positive, neutral, or purifying selection. 6. Why is fitness important in evolution? How does it relate to selection? Fitness increases the probability of producing offspring that can survive in an environment; if a genetic variant increases fitness, this is probably going to be selected for. 7. Genetic variants come in multiple flavors. What are they and how do they impact fitness and selection? How do they lead to evolution? Genetic variants can either be positive, neutral or negative. Positive leads to increased fitness; neutral has no effect on fitness; negative means decrease in fitness. They can either be selected for or against, or can be neutrally selected. These changes and the variants being selected for/against leads to evolution on a micro scale in large part due to the neutral variants being inherited/being “side effects” of traits that are selected for. 8. What is an example of natural selection during dog domestication? Artificial selection? The appearance of domestication behaviors is an example of natural selection. It arose from random genetic variation in wild population. An example of artificial selection is modern dog breeds and enhanced morphological variation that is driven by highly selective breeding. 9. What kinds of tools can we use to describe dog (or other species) evolution? Tools that can be used to describe evolution are copy number variance, genome sequencing, structural variants, and genome sequencing. 10. What sort of genetic variants can be informative when studying evolution? How are CNVs related to animal evolution? Structural variants? Every genetic variant is useful except the purifying genes in studying evolution. CNVs show the difference in genetics that contribute to evolution, such as dogs being able to digest starch contributed to their domestication and formation as their own species. Structural variants result in changes within the genome that can effect things like appearance and behavior, which can be selected for. Lecture 19 Extra effort: How would you go about discovering the genes that have been selected for or against during the last 200 years of dog breeding? Imagine you are interested in the genes for skull shape in Bull Terriers (or another specific example). Over the past 200 years, variations in the phenotypes have been selected for that lead to changes in things like the skull shape of bull terriers. I would use a GWAS to analyze bull terriers and compare them to dog breeds without the football shaped head, which would be the control. The genotype of the bull terrier would be compared with the controls to ID regions of the genome that may potentially be related to the skull shape.

Exam 4 Review

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