MBG 3350 - Term Test 2 Questions with Correct Answers Latest Update 2025/2026

MBG 3350 - Term Test 2 Questions with Correct Answers Latest Update 2025/2026 Why is it typically necessary to use fewer than 30 cycles when making quantitative comparisons using end-point PCR? - Answers After 30 cycles, most reactions will have reached the plateau phase and the amount of amplified product will no longer accurately represent the amount of target sequence in the original sample What is the specificity of SYBR-Green? - Answers Detects all amplified dsDNA What is the specificity of TaqMan or Beacon probes? - Answers Detects specific amplification products only What are the advantages of using TaqMan or Beacon probes? - Answers low background, low risk of false positives, can label different probes with different dyes (multiplexing) and high level of quantification for gene expression What are the advantages of using SYBR-Green? - Answers No probes required, simple set up and low cost What are the disadvantages of using SYBR-Green? - Answers greater risk of false positives, no multiplexing possible What are the disadvantages of using TaqMan or Beacon probes? - Answers different oligo probes needed for each target sequence How does TaqMan qPCR work? - Answers TaqMan probe binds to template just downstream of one primer (third oligonucleotide). The probe has a flurophore is on the 5' end and a quencher on the 3' end. Fluorescence is quenced and no fluorescence is detected as long as the quencher is in close proximity to the fluorophore. During polymerization, Taq poly (which has 5'-3' exonuclease activity) displaces and cleaves the fluorophore labeled 5' nucleotide of the probe. This releases free reporter due from the probe which now fluoresces since it is no longer quenched. The fluorescent signal is detected and recorded after each cycle. What two things can you do to ensure your primers are not binding non-specifically to other targets within the genome? - Answers The reaction can be 1) separated on an agarose gel to view the amplified products or 2) a melt curve analysis can be done to determine the specificity of the primers Describe the steps taken to prevent/reduce genomic DNA contamination in RNA preparations used for RT-PCR - Answers Extract RNA under acidic conditions, treat with RNase-free DNase Describe strategies of primer design to reduce the chance of amplifying specific products from genomic DNA - Answers Design primers that either cross an exon-intron boundary or that are in different exons Describe a PCR control that would confirm the above strategies were successful - Answers "minus RT" control, where a sample of the RNA that has not been reverse-transcribed is added to the PCR with primers for a gene of interest Why is a preparation of RNA treated with RNase-free DNase and why is that done before the reverse transcription step? - Answers DNA must be removed from an RNA preparation so that the DNA can make no contribution to the amplicon. This must be done prior to the RT step because the first strand cDNA after reverse transcription would be susceptible to DNase digestion.