TAMU BIO 111 LAB EXAM 2 REVIEW QUESTIONS AND ANSWERS 2024 LATEST UPDATE.

What is the difference between the coding and non-coding region? Coding region: comprised of genes that encode proteins (instructions that determine traits) and makes up 1-2% of human DNA. • Noncoding region: makes up 98-99% of human DNA, contains STRs and is responsible for regulatory functions such as gene transcription. What are STRs? Short-tandem repeats (STRs) are unique repeating patterns of the same nucleotide sequence. They can be used to differentiate one person from another. Brainpower Read More Previous Play Next Rewind 10 seconds Move forward 10 seconds Unmute 0:00 / 0:15 Full screen What is a DNA profile? A DNA profile is a specific pattern of DNA attributes that is obtained in the lab and can be used to identify an individual. • DNA profiling is the process of determining an individual's DNA characteristics (profile) from a sample of bodily tissue What does PCR stand for? What is the purpose of PCR? Polymerase Chain Reaction • Purpose is to amplify DNA. In forensics, this can be used to obtain more DNA from a sample that would otherwise be too small to analyze. What is inside the master mix? 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 the chelex do? The chelex resin chelates (absorbs) ions that inhibit function of the Taq polymerase enzyme What does the thermocycler do? Thermocyclers are instruments used to amplify DNA and RNA samples by the polymerase chain reaction. The thermocycler raises and lowers the temperature of the samples in a holding block in discrete, pre- programmed steps, allowing for denaturation and reannealing of samples with various reagents. What are the 3 steps of a PCR cycle? What happens during each step? • 1) Denaturation, 2) Annealing, 3) Extension • Denaturation: When the double stranded template DNA is heated to separate it into two single strands • Annealing: when the temperature is lowered to enable the DNA primers to attach to the template strands • Extension: 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 What is the purpose of gel electrophoresis? Allows you to separate a mixture of different sized DNA molecules so that DNA samples can be compared to other known DNA profiles. What is the charge of DNA? DNA has a negative charge What direction should DNA run? Toward the positively charged end (anode) What are things that influence how far DNA will run? Charge of molecule: DNA carries a negative charge so attracted to positive end • Size: Smaller molecules/fragments will travel faster and further • Density of gel: the more dense the gel is, the slower the particles will move. You want a gel that is dense enough to separate particles but that won't take too long Who will run further from a well? 700bp of DNA or 200bp of DNA. 200 bp (smaller, so travels faster and further) Why is developing a DNA profile important? DNA testing allows forensic investigators to identify a guilty individual with a high degree of certainty because the DNA sequence of every person is unique (except for identical twins) How did multiplex PCR help in our analysis versus just using a single pair of primers? A single pair would not allow you to distinguish between samples/individuals with a high degree of certainty Why are DNA ladders (standards) important? They are used to determine the sizes of bands in other DNA samples. Thus, we are able to compare them. Can you tell the difference between a homozygote or heterozygote gene on a gel? Yes, a homozygote will have one band at a particular loci and a heterozygote will have two bands at a particular loci What was the purpose of using SYBR Green in the gels? SYBR Green binds to DNA and fluoresces when exposed to the right wavelength of light. This makes the separated bands visible. What was the purpose of pouring the buffer in the electrophoresis chamber? Buffer controls the net charge of molecules by maintaining the pH at nearly neutral. This facilitates proper migration and separation of DNA molecules. What is the central dogma of gene expression? Define transcription and translation. The central dogma states that genes specify the sequence of mRNAs, which in turn specify the sequence of amino acids making up all proteins o Transcription: DNA from a gene sequence is copied to mRNA o Translation: directs the synthesis of proteins What is the name of the primary enzyme used in this lab? Where is it coming from? B-galactosidase is an enzyme is E. coli that is encoded by the LacZ gene when E. coli needs to break down lactose What are the components of the lac operon and what do they do? Operon: is a functioning unit of DNA containing a cluster of genes under the o RNA polymerase: needed to start transcription and produce mRNA o Promoter: the binding site for RNA polymerase o Operator: negative regulatory site that the repressor can bind to o CAP site: positive regulatory site that catabolite activator protein (CAP) can bind to o Repressor: when the repressor is bound it blocks RNA polymerase and prevents the production of mRNA (so no production of proteins) o The lac operon has 3 genes that help the cell utilize lactose: 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 E. coli makes B-gal to breakdown what sugar? Lactose 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? Because B-gal also breaks down ONPG and one of the products of this reaction has a yellow color (ortho-nitrophenol), we can investigate the regulation of LacZ transcription by observing the rate of color change in the tubes. Presence of yellow color would mean that B-gal was produced, thus LacZ would be expressed.