BIO lab exam 2 TAMU (2023/2024) Rated A+

BIO lab exam 2 TAMU (2023/2024) Rated A+ Coding regions sequences of DNA that serve as instructions for making proteins (1-2% of human DNA) Non-coding regions Human DNA that contains STRS and are responsible for regulatory functions ( DNA transcription 98-99% ) What are STRs? short tandem repeats; unique repetition of nucleotide patterns. Differentiate one person from another. What is a DNA profile? a specific pattern of DNA attributes that is obtained in the lab and can be used to identify an individual; is the process of determining an individual's DNA characteristics (profile) from a sample of bodily tissue What does PCR stand for? polymerase chain reaction What is the purpose of PCR? to amplify a specific desired fragment of DNA (make more copies) What is inside the master mix (forensic biology I)? 3 pairs of primers, loading dye, nucleotides (dNTPs), Taq polymerase, mix buffer, and water. DNA sample is added to master mix. What are the components of PCR? and what do they do? • Template: the DNA from the sample specimen serves as the template for replication • Primers: short stretches of DNA that initiate the PCR reaction. • Required for annealing and Taq binding at specific sites • Base sequences are complementary to the ends of the template DNA • Taq polymerase: adds dNTPs to the template strand • Reads the original DNA sequence and creates a complimentary copy by adding in the new DNA bases • Nucleotides (dNTPs): DNA bases (A, C, G, and T) serve as the building blocks of DNA and are used to assemble new strands of DNA • Buffer: enables the reaction to take place by ensuring the right conditions are met (controls pH) What does Chelex do? The chelex resin chelates (absorbs) ions that inhibit function of the Taq polymerase enzyme What does the thermocycler do? instruments used to amplify DNA and RNA samples by the polymerase chain reaction. It raises and lowers the temperature of the samples in a holding block in discrete, allowing for denaturation and re-annealing of samples with various reagents. 3 steps of PCR cycle 1. denaturation 2. annealing 3. extension Denaturation (PCR) When the double stranded template DNA is heated to separate it into two single strands Annealing (PCR) when the temperature is lowered to enable the DNA primers to attach to the template strands Extension (PCR) when the temperature is raised and the Taq polymerase enzyme synthesizes new DNA strands by adding nucleotides to the template strands. What is the purpose of lysing the DNA? Lysis destroys the cell membrane and releases the contents of the cell into the solution. what organism was Taq polymerase isolated from? Thermus aquaticus: thermophilic bacterium (heat tolerant) that live in hot springs What is multiplex PCR? Amplification of multiple targets into a single PCR experiment by utilizing multiple primer pairs in a single reaction mixture Summarize what happens in electrophoresis 1. Agarose and buffer solution are put into a plastic tray. A comb is placed to make wells at one end. 2. DNA samples colored with a tracking dye are pipetted into the wells. 3. Tray is placed into a chamber that generates electric currents through the gel. 4. The DNA has a negative charge making it go to the positive side (anode); the smaller DNA molecules will move the fastest through the gel. - DNA ladder will contain DNA fragments of known sizes. purpose of gel electrophoresis allows you to separate a mixture of different sized DNA molecules so it can be compared to other known DNA profiles. Things that influence how far DNA will run? -Charge of molecule -Size -Density of gel [ too dense makes it take longer] How did multiplex PCR help in our analysis versus just using a single pair of primers? Using a single pair would not distinguish with a high degree of certainty. Why are DNA ladders important? determine the sizes of bands in other DNA samples Homozygote gene one band at a particular loci Heterozygote gene two bands at a particular loci The purpose of using SYBR green in the gels? Binds to DNA and fluoresces when exposed to the right wavelength Purpose of using the buffer in the electrophoresis chamber? It controls the net charge of molecules by maintaining the PH neutral central dogma of gene expression Genes specify the sequence of mRNA which specify the sequence of amino acids making up all proteins Transcription DNA is copied into mRNA Translation directs the synthesis of proteins Primary enzyme used in gene expression & regulation lab? B-galactosidase is an enzyme in E. coli that is encoded by the LacZ gene when E. coli needs to break down lactose Operator negative regulatory site that the repressor can bind to CAP site Positive regulation-site for catabolite activator protein (CAP) can bind to Lac operon genes lacZ - encodes β-galactosidase enzyme, which splits lactose into monosaccharides lacY - encodes lactose permease protein, which is a transmembrane "pump" that allows the cell to import lactose lacA - encodes transacetylase enzyme Expression of the LacZ gene causes the production of what enzyme? B-gal How can ONPG be used to measure optimal growth conditions for the expression of LacZ? Presence of yellow color would mean that B-gal was produced, thus LacZ would be expressed. LacZ expression is high in what culture? LB+lactose culture How to calculate activity Absorbance/time*1000 What sugars are the products of the hydrolysis of lactose? Galactose and glucose When is the repressor bound or unbound to the operon? The repressor is bound to the operator when lactose is absent. When lactose is present the repressor is prevented from binding. In order to make more B-gal, what will the repressor bind? Allolactose would have to bind to the repressor How to calculate total magnification Objective magnification*10X = total magnification How to use a compound microscope 4X objective lens and focus with the coarse adjustment knob, move to 10X lens and use the fine adjustment knob, then move to 40X and refocus again with the fine adjustment knob Number of chromosomes at each stage of interphase and mitosis 46 chromosomes in interphase, prophase and metaphase. 92 chromsomes in anaphase and telophase and back to 46 after cytokinesis. Mitosis produces 2 genetically identical daughter cells and conserves the number of chromosome numbers. Genetic variation does not change. Meiosis occurs in sexually reproducing organisms and results in cells with half the chromosome number of parent cells. Produces 4 haploid daughter cells and increase genetic variation. Why did we only want to look at the root tips to observe the cell cycle? Apical meristem is located in root tips. Region of rapid progression through the cell cycle. What plant mutants were worked with in class? What are their phenotypes? We worked with Arabidopsis thaliana, which is a small weed in the mustard family and is a common model organism for studying plants -One parent had trichomes and fluoresced (hair + glow) -One parent was glabrous and did not fluoresce (no hair + no glow) Phenotype the physical characteristics of a trait Genotype The particular combination of alleles for a gene or locus (EX. AA, Aa) Homozygous two identical genes for the same trait (AA or aa) Heterozygous two different genes for the same trait (Aa/Bb) Gregor Mendel father of modern genetics Mendel's Laws law of dominance, law of segregation, law of independent assortment Law of dominance two organisms that are each homozygous for two opposing traits are crossed, the offspring will be hybrid [ 2 different alleles] but will exhibit only the dominant trait Law of Segregation during the formation of gametes, the two traits carried by each parent will separate law of independent assortment states that during gamete formation, the alleles for one trait, segregate independently from the alleles of a gene for another trait. Monohybrid cross (Tt x Tt) two organisms that are each hybrid for one trait. Ratio- 1:2:1 Dihybrid Cross (TtYy x TtYy) cross between individuals that are hybrid for two different traits. Can produce 4 different types of gametes. Ratio- 9:3:3:1 What is bioinformatics? application of computer technology and associated software to biological data NCBI National Center for Biotechnology Information What is NCBI used for? Is a database that stores sequence information, taxonomic data, 3D protein structures, and a vast array of online and offline software tools. What does BLAST stand for? Basic Local Alignment Search Tool What is BLAST used for? used to generate alignments between a nucleotide or protein sequence between other sequences in the database Difference between BLASTn and BLASTp? -BLASTn compares nucleotide sequences -BLASTp compares protein sequences Purpose of phylogeny? To group organisms by similarity (i.e., species, genes, proteins, morphology, etc.) What is the max score? based on the length of the query sequence matched. It is the highest alignment score calculated from the sum of matched and mismatched nucleotides or amino acids. Percent sequence identity tells you how many characters (bases or amino acids) match exactly between the query sequence and the target sequence E-value Represents the probability that you would get a match with that score by random chance (the number of expected hits of similar quality that could be found by chance)