PCR and RT-PCR QUESTIONS WITH
ACCURATE ANSWERS
Additional assays, such as ___________________ , may be performed after RT-PCR to identify non-
specific amplification. correct answer melt curve analysis
Annealing Temp is based on the _______________________ of the ______________ , where Tm is the
temp at which ________% of the primer binds specifically to the template. correct answer melting
temperature (Tm) - primer - 50%
At what phase of Real-Time PCR do reaction components become limited and the reaction stops?
correct answer Plateau phase
At what phase of Real-Time PCR does the reaction slow down due to depletion of reagent components
and lower PCR efficiencies. correct answer Log-linear phase
At what phase of Real-Time PCR must quantification be done? correct answer Early exponential phase
At what step is the fluorescence signal observed? correct answer annealing step
Ct directly or inversely correlates with the amount of template in the sample. correct answer inversely
Define "Ct". correct answer Ct (threshold cycle): the cycle number at which the fluorescence signal
crosses the threshold.
Describe how FRET probes are used for detection. correct answer 2 oligonucleotides bind to the PCR
product in a sequence-specific fashion. When 2 fluorescence resonance energy transfer (FRET) probes
bind to the targets in close proximity, the energy transfer from the donor fluorophore to the acceptor
fluorophore takes place allowing emission of fluorescence from the acceptor. The fluorescence signal is
observed during the annealing step. One probe has a donor dye at the 3' end and the other probe has an
acceptor on the 5' end.
Describe the typical standard PCR Protocol. correct answer 1. Initial denaturation
