Type: Knowledge | Weight: 19% of Exam
Focuses on the theoretical understanding of common molecular biology assays and the ability to select the correct assay for a given scenario.
Key Topics:
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In a conventional PCR reaction, what is the primary function of the step usually performed at 72∘C?
Extension of the new DNA strand by Taq polymerase
The step at 72∘C is the extension (or elongation) step, where Taq polymerase synthesizes the new DNA strand complementary to the DNA template. Denaturation occurs at ≈95∘C and annealing at ≈50–65∘C.
Which of the following distinguishes Real-Time PCR (qPCR) from conventional PCR?
qPCR allows for the quantification of DNA accumulation during the exponential phase.
qPCR (Quantitative PCR) measures the accumulation of DNA in real-time during the exponential phase using fluorescence, allowing for quantification. Conventional PCR relies on end-point detection using gel electrophoresis.
In a SYBR Green qPCR assay, you observe a specific melting curve with a single peak. What does this indicate?
A single specific amplicon was generated.
In melt curve analysis, a single peak indicates that a single specific DNA product (amplicon) with a specific melting temperature (Tm) was amplified. Multiple peaks would suggest non-specific binding or primer-dimers.
If sample A has a Ct value of 20 and sample B has a Ct value of 23 in a qPCR experiment, what is the relative difference in starting template quantity, assuming 100% efficiency?
Sample A has 8 times more DNA than Sample B.
The difference in Ct is 23−20=3. Assuming 100% efficiency (doubling every cycle), the fold difference is 2ΔCt=23=8. Since Sample A has the lower Ct, it started with more template.
Which component is required for Reverse Transcription PCR (RT-PCR) that is not used in standard genomic DNA PCR?
Reverse Transcriptase
RT-PCR starts with RNA. Reverse Transcriptase is the enzyme required to convert RNA into complementary DNA (cDNA) before standard PCR amplification can occur.
When designing primers for PCR, which of the following characteristics is most desirable to prevent non-specific binding?
Unique sequence specificity to the target region and absence of secondary structures.
Primers should be specific to the target and lack secondary structures (like hairpins). Complementary 3' ends cause primer-dimers. Tm should be similar, and GC content ideally 40-60%.
What is the role of Mg2+ ions (usually from MgCl2) in a PCR reaction mixture?
It acts as a cofactor for Taq polymerase activity.
Mg2+ is a cofactor for DNA polymerase. It affects enzyme activity, primer annealing, and the melting temperature of the DNA.
TaqMan probes differ from SYBR Green in qPCR because:
TaqMan relies on a fluorophore-quencher pair on a specific oligonucleotide probe.
SYBR Green binds any dsDNA. TaqMan uses a specific probe labeled with a fluorophore and a quencher; fluorescence is released only when the polymerase cleaves the probe during extension.
Identify the issue in this PCR gel result: A smear is observed in the negative control lane (water only).
There is contamination in the reagents.
The negative control should have no bands or smears. Presence of DNA in the water control indicates contamination of reagents or pipettes with foreign DNA or amplicon.
Which formula calculates the annealing temperature (Ta) often used as a starting point, relative to the primer melting temperature (Tm)?
Ta=Tm−5∘C
A common rule of thumb for starting annealing temperature is approximately 3–5∘C below the lowest Tm of the primer pair.
