PCR Flashcards
Prior to PCR, Southern Blotting was a commonly used protocol to detect specific DNA sequences. Laboratory investigators undertaking this technique were required to:
All of the above
Taq polymerase from Thermos aquaticus has properties that make it ideal for PCR. These properties include:
Some of the above are properties that are useful for PCR
Why is Escherichia coli DNA polymerase not very useful for PCR?
E. coli DNA polymerase is heat labile
True or false: the two primers that are used to target a region for amplification are themselves incorporated into their resulting amplicon.
True
In a single PCR cycle consisting of 30 seconds at 95 degrees, one minute at 55 degrees and one minute at 72 degrees, what is happening in the step run at 55 degrees?
Primers are annealing to the DNA to be amplified
Taq polymerase is a:
DNA-dependent DNA polymerase
Prior to PCR, Southern Blotting was a commonly used protocol to detect specific DNA sequences. The DNA and the gel was blotted onto:
Nitrocellulose
Which component of the PCR reaction mixture confers specificity in the reaction for the target sequence of the original DNA template?
Primers
Prior to PCR, Southern Blotting was a commonly used protocol to detect specific DNA sequences. The developed X-ray post-blotting and storage at -80C is referred to as:
Autorad
Prior to PCR, Southern Blotting was a commonly used protocol to detect DNA sequences. It required the use of radioactive nucleotides to be included in the probe; this often consisted of:
Radioactive phosphorus
A hallmark of the enzymes used to carry out PCR is:
Their ability to survive repeated exposure to high temperature
All of the following are required components of PCR except:
All are required
Polymerase chain reaction was developed to amplify small segments of DNA; which of the following samples cannot be used in a PCR reaction?
All can be used
True or false: a short number of cycles and PCR, such as 20, will not yield sufficient applicants for gel analysis.
True
During which step of PCR does synthesis occur?
Elongation
In PCR, the amplicon increases ______________ with each cycle.
Exponentially
Why must DNA be denatured during PCR?
Opens up the double helix and allows primers to bind to complementary sequences
Which statement about Thermos aquaticus is false?
Found in the frozen lakes of Antarctica
An addition to DNA polymerase a PCR reaction mixture contains all of the following, except:
Ligase
Which of the following is the correct order of the steps that occur in a standard PCR cycle?
Denaturation, annealing, and extension
Reverse transcriptase PCR is useful for using mRNA with introns removed in order to obtain:
cDNA
RT-PCR refers to:
Reverse transcriptase PCR
Why would a researcher want to use RT-PCR?
RT-PCR generates a DNA molecule without the noncoding introns from eukaryotic mRNA
True or false: the 3’ end of a primer must be fully complementary to a sequence in order for PCR amplification to take place.
True
True or false: a PCR master mix can be divided into aliquots but they should never be refrozen.
False
What DNA sequence information is essential for the success of PCR?
The sequence of the ends of the DNA to be amplified must be known
Ideally, the TM values for a primer pair should be within:
5 degrees of each other
What type of PCR starts with an RNA template?
RT-PCR
here is a target sequence in single strand format. What is the sequence of the reverse primer, written in the 5’ to 3’ direction?
GGTATGACATATAACGTA
Primer pairs should have compatible melting temperatures preferably within a maximum difference of:
50 degrees from one another
True or false: primer annealing temperature is synonymous with primer melting temperature.
False
All of the following are required components of PCR except:
DNA gyrase to unwind the DNA
Which technique would be best suited to determine whether a specific gene is being expressed in a tissue sample?
Northern blotting
Setting a thermal cycler to a specific primer TM value across one row and using this as a mean temperature, whereby the remaining rows have stepwise variations from higher to lower than the mean temperature is called:
Gradient temperature PCR
RT- PCR is basically a four step process: 1. ____________,2. reverse transcription, 3. PCR amplification, and 4. PCR product analysis.
RNA isolation
Given a drug influenced the CT value of 29 for expression of Gene MLS3 after 24 hours, and a CT value of 34 at time zero, what is the fold increase/decrease for this drug at time 24 hours, compared to time 0? Expression of a control gene generated CT values of 14 at time zero and 16 at 24 hours respectively.
128
Real-Time PCR differs from standard PCR and which way?
Real-Time PCR is quantitative
The reporter molecule on the Taqman probe is found:
At the 5’ of the probe
In qPCR, the quencher molecule:
Might be a fluorophore
The reporter molecule of the Taqman system:
Fluoresceces constantly, irrespective of whether amplification is taking place or not
True or false: RT PCR and qPCR are synonymous terms that refer to real time PCR.
False
In a real time PCR run of two samples, Sample A has a CT value of 20m while Sample B has a CT value of 17; this suggests that:
Sample A has eight-fold less target sequence than Sample B
A direct correlation exists between template copy number and the sample vial pre-PCR and the early cycles of the ______________ phase.
Exponential
In qPCR, two peaks observed following melt curve analysis indicate:
Two regions of DNA have been amplified and they are being dependent on their melt temperature.
Real time PCR can be used to determine gene duplications or deletions. For example, a normal individual carries two copies, one paternal and one maternal, for the red gene and also two copies for the green gene. However in another individual, it has been determined that one copy of the red gene has been deleted. Which of the two real time PCR screen readouts is the correct readout for the red gene deletion?
A
qPCR provides the following result: Sample A has a CT value of 22, Sample B has a CT value of 21, and Sample C has a CT value of 26. Following the completion of the qPCR run:
Sample C has the lowest amount of starting amplicon at the beginning of the Q PCR assay
RN refers to normalized reporter fluorescence for FM; reporter RN is determined by:
Dividing reporter fluorescence value by ROX fluorescence value
Retroviral RNA-directed DNA polymerase is better known as:
Reverse transcriptase
ROX:
Is a normalizer fluorophore
The filter sets that are included in real time PCR instruments to ensure that the optimal wavelengths of light for sending to the fluorophore and for the optimal signal detection are referred to as the:
Excitation and emission filter sets
The plateau phase is:
Not important in qPCR assays
What does ‘P’ represent in the formula A = Ao x Pn?
Efficiency of the PCR reaction
When PCR efficiency is low, the PCR amplicon:
May not form a visible band on a gel.
What does ‘Ao’ represent in the PCR efficiency formula?
Starting amount of amplicon prior to PCR
A single nucleotide weighs:
330 daltons
A bp is a loose term that molecular biologists use to describe a:
Nucleotide on one strand of double-stranded DNA that is hydrogen-bonded to a nucleotide on the complementary strand
If a single molecule of water weighs 18 daltons, then a mole of water weighs:
18 grams
What does ‘A’ represent and the formula for PCR efficiency?
Yield after PCR
The molecular weight of an individual atom or an individual molecule is described using which weight unit?
Dalton
What does ‘n’ represent in the PCR efficiency formula?
Number of cycles
