Genetics Lab Final Exam Study Guide Fall '24 PDF

Genetics lab final exam study guide Fall ‘24 I would recommend that in addition to reviewing all of the powerpoints and lab notebooks that you make sure that you understand everything in this guide. The exam will consist of 30-35 questions and will be a combination of multiple choice, true/false, an...

Genetics lab final exam study guide Fall ‘24 I would recommend that in addition to reviewing all of the powerpoints and lab notebooks that you make sure that you understand everything in this guide. The exam will consist of 30-35 questions and will be a combination of multiple choice, true/false, and written response questions. The exam will test both theory and practical applications of the course material. Students are expected to be able to: 1. Explain homology and how it allows for the development of model organisms 2. Identify homologous genes in different organisms given a gene sequence 3. Identify sgRNAs with an adjacent PAM domain 4. Explain whether an in-vitro CRISPR experiment worked and identify potential causes of its failure. 5. Understand how to interpret a gel for experiments. 6. Implement correct pipetting techniques while using a micropipettor Module 1 Mosquitoes are animals in the order Diptera. The have a close relationship with water, requiring water for 2/3 of their life span. In this class, we focused on the mosquito Aedes aegypti, a common vector for dengue and Zika virus in South Florida, as a model organism to study mosquito hydrosensation. Based on data from another Dipteran, the fruit fly Drosophila melanogaster, we are interested in two genes of interest that we may be involved in sensing water: Ionotrophic Receptor 68a (IR68a) and Odorant Binding Protein 71 (OBP71). We can study these genes in one organism to make inferences about their role in another because of homology, i.e. their similarity due to shared ancestry. Everything we’ve done in this course has been in the service of preparing for and simulating a reverse genetics experiment. Reverse genetics is when a mutant genotype is made and the phenotype of that mutant is assessed. Compare this to forward genetics, a more traditional approach in which a mutant phenotype is observed and the locus causing that phenotype is determined by mapping. In this module, we discussed how to use a 2 x 2 factorial design for reverse genetics experiments. In short, one independent variable will always be (in this class) genetic background (i.e., WT or mutant), and the other will be some treatment that you expect the genotypes may respond to differently. The dependent variable, then, will be some factor that you measure across your four treatments. Two of these four treatment groups will be your positive control (ensures that the experiment works; protects against false negatives) and your negative control (shows you what “no treatment” looks like; protects against false positives). Module 2 Understand, basically how to use a pipette: specifically, the volumes that each tool is calibrated to use and how to read the volume dial. Understand the relationship between volumes that use difference metric prefixes (in this class we live primarily in the micro-/u to milli-/m range). Remember, a unitless answer is always wrong! Module 3 Understand the principle that gene families are created by duplication within a genome to create paralogs as well as speciation events to create orthologs; there is a specific diagram in the slideshow you can use to review this. Understand that both of these are types of homologs and that these are technically different from analogs. BLAST stands for “basic local alignment search tool.” Understand the different types of BLAST that we used in class and how it scores sequences based on similarity. Understand the outputs like e-value, query cover and percent identity. An exon is a segment of a gene that codes for a protein and that an intron is a segment of a gene that does not. All of the exons together combine to form the CDS, or coding DNA sequence of a gene. At the 5’ and 3’ end of a gene there may be UTRs, or untranslated regions. UTRs, exons and introns are all transcribed into pre-mRNA, the pre-mRNA is then processed into mRNA by cleaving out the introns and UTR leaving a transcript comprised only of the exons, and that mRNA is translated into protein. Be able to understand a gene map (diagram showing exons and intron gaps). Be able to recognize a FASTA file and tell whether it displays nucleotides or amino acids. Be able to translate a nucleotide sequence using a codon chart; remember, when DNA is transcribed into RNA the Ts are replaced with Us. Module 4 Understand the basic principles of column-based extraction techniques, notably that we need to access and purify DNA by lysing cellular and nuclear membranes as well as removing proteins and small molecule metabolites. Remember the steps to DNA extraction are: Homogenization (manual breakup of the specimen into smaller chunks), Cell and Nuclear Lysis (we breakup the cell and nuclear membranes and dump the contents of it into solution), Binding of DNA (we bind our DNA to a filter or beads), Washing (wash away proteins and inorganic contaminants using Ethanol washes), and Elution (we release the DNA from the filter/beads and elute it into our final liquid medium). Remember the role of detergents in cell membrane lysis and the role of EDTA in protecting DNA from enzymatic degradation. *Study the role of each of the reagents used in the extraction, see DNA extraction module* Module 5 Generally, genotyping of mutations is going to start with amplification of the region including the target site. This is most simply accomplished by a procedure known as PCR, or polymerase chain reaction. PCR consists of three steps that constitute one cycle, and is repeated for many cycles. At the end of each cycle, there is up to twice as much of the target sequence or amplicon as was present at the beginning of the cycle. A cycle consists of a denaturation step, in which heat is used to break the hydrogen bonds holding the two sides of the DNA “ladder” together, creating two complementary single-stranded DNA molecules. The next step is annealing in which the temperature is lowered and two short, single-stranded primers are able to bind to their complementary targets in the sample. The final step is elongation in which an enzyme known as Taq polymerase binds to the sample DNA/primer complex and elongates the complementary strand using dNTPs (essentially loose nucleotides) from the solution. The use of Taq polymerase represents an innovation because it is from a thermophilic prokaryote and thus able to operate at a temperature in which your sample DNA strands are naturally separated. Modules 6, 7 Once you have successfully amplified your region of interest, you can investigate its size using some technique like gel electrophoresis. Gel electrophoresis is the process of using an electric current to separate DNA products by size (length is measured in basepairs) as they move through an agarose matrix; DNA is negatively charged and will move away from an anode, and how far it is able to migrate through the gel during a given time is inversely proportional to its size. Gel bands are compared to a set of standard values like a DNA ladder. Note that gel electrophoresis is not efficient for detecting very small indels. In order to do that, you can use a process like fragment analysis, which is essentially the same process but using a capillary instead of the gel and creates a digital readout. Alternatively, you can perform a restriction digest when relevant. Restriction enzymes cut DNA at specific sites; if one of these sites overlaps your CRISPR target you can create mutations such that the wild type will be cut by the enzyme but the recognition site for the restriction enzyme is disrupted by the mutant and will result in no cutting. You should be able to interpret such a gel by looking at it. To purify our samples before sequencing, we used ExoSAP, a product containing exonuclease I and shrimp alkaline phosphatase. ExoI cleaves unconsumed primers in our reaction into dNTPs and SAP renders dNTPs into unusable nucleosides and inorganic phosphate. Together, these two enzymes remove components that can interfere with downstream reactions. To determine the sequences of our amplified fragments we used Sanger aka cycle sequencing. Understand the basics of the cycle/Sanger sequencing reaction, specifically how it is similar to and different from PCR (i.e., use of only one primer, use of labelled ddNTPs). REVIEW: Module IV: DNA Electrophoresis Lab Notebook Module 8 Understand how to read a Sanger sequencing gel electrophoresis (see example X). Know what a polymorphism is. CRISPR stands for clustered regularly interspersed palindromic repeats. This name has little to do with how the technology is used and more to do with the genomic elements related to it in the bacteria in which it was discovered. Cas9 stands for CRISPR associated protein 9; other Cas proteins also cut DNA. Cas9 and its predecessors like TALENs and ZFNs are nucleases, enzymes that cleave DNA. The difference between random mutagenesis and genome editing is most simply that genome editing results in specific, targeted mutations. Cas9 can cut DNA anywhere there is a PAM, or protospacer adjacent motif. A PAM takes the form “NGG” in the genome, where “N” is any nucleotide. The 20 bases 5’ of the PAM are the target sequence and determine the specificity of your edit. The PAM is not part of the target. Cas9 will cut ~4-5 bases 5’ of the PAM. CRISPR-Cas9 genome editing results most commonly in a small insertion or deletion (or indel) resulting from faulty repair during a process known as NHEJ or non-homologous end joining. Typically, we are interested in creating a knockout mutant, or one in which the effect of the mutated gene is negligible or zero. The simplest way to do this is to create a truncated protein. Indels that insert or remove a number of bases that are not a multiple of three will create a frameshift mutation—one in which the reading frame of the codons is disrupted. When a frameshift results in a change in codons, that is known as a missense mutation (think back also to the codon chart and central dogma slides that have been peppered in as early as Module 3). When a frameshift results in a stop codon, that is known as a nonsense mutation. A frameshift will typically result in some amount of missense after the indel and ultimately an early stop codon as well. Example Sanger Sequencing Gel: