PCR Quiz
Questions: 16 · 10 minutes
1. After lowering the annealing temperature substantially, a reaction produces several extra bands. What is the most likely explanation?
The DNA polymerase can no longer extend any primer
The no-template control has become a positive control
The template DNA has been converted into RNA
Primers are binding less specifically to unintended sites
2. Why is an end-point band from conventional PCR generally unsuitable for precise measurement of starting template quantity?
DNA cannot be visualized after amplification
Primer sequences always change during the final cycle
Conventional PCR uses RNA polymerase rather than DNA polymerase
Reactions may reach a plateau where final product no longer reflects the initial amount proportionally
3. In reverse-transcription PCR designed to detect an RNA target, what happens before or as amplification begins?
The primers are translated into enzymes
The RNA is separated into proteins by heat
The RNA is converted into complementary DNA
The DNA polymerase converts the target directly into lipids
4. Why is a thermostable DNA polymerase commonly used in PCR?
It makes primers unnecessary
It remains functional through repeated high-temperature cycles
It allows amplification to occur without nucleotides
It permanently joins the two template strands
5. Which workflow most directly reduces contamination of new PCR reactions by previously amplified products?
Opening amplified-product tubes beside the clean reaction setup area
Physically separating pre-PCR setup from post-PCR work and using dedicated equipment
Moving repeatedly between post-amplification and pre-amplification areas while setting up reactions
Using the same pipette for post-PCR analysis and master-mix preparation
6. What is the main purpose of a standard PCR reaction?
To amplify a selected DNA region
To separate proteins according to size
To translate messenger RNA into protein
To determine every base in a genome directly
7. The positive control does not amplify, and none of the unknown samples amplify. What is the best conclusion?
The run may have failed, so negative sample results cannot yet be trusted
Every unknown sample is confirmed negative
The unknown samples must all contain PCR inhibitors
The target sequence is absent from the positive control by definition
8. Two samples are tested by the same efficient qPCR assay under comparable conditions. Sample A crosses the fluorescence threshold earlier than Sample B. What does this usually indicate?
Sample A contained more starting target sequence
Sample B necessarily contains contamination
Sample A has a longer amplicon than Sample B
Sample A contains no amplifiable target
9. A no-template control produces a band matching the expected amplicon size. What is the most likely interpretation?
The positive control contained too little loading dye
The tested samples contain unusually large amounts of target DNA
The gel ran in the wrong direction
Target DNA or amplified product contaminated the control or reaction mix
10. A PCR mix contains substantially more magnesium ions than the assay was optimized to use. What can happen?
All DNA is automatically degraded before cycling
Specificity may decrease, allowing more nonspecific amplification
Primers can no longer contain nucleotide bases
The reaction becomes an RNA-sequencing assay
11. An extraction blank amplifies, but the no-template control prepared from clean PCR reagents does not. Where was contamination most likely introduced?
During extraction or handling of extracted material
Only within the unknown samples before collection
During gel imaging after electrophoresis
By the absence of DNA in the no-template control
12. Which mixture best describes a no-template control for PCR?
Template DNA and polymerase, but no primers
All PCR reagents, with template replaced by nuclease-free water or buffer
A known target-positive sample processed with the unknowns
Template DNA and primers, but no nucleotides
13. What primarily determines the boundaries of the DNA region amplified by PCR?
The concentration of loading dye
The volume of the reaction tube
The binding locations of the forward and reverse primers
The migration direction used during gel electrophoresis
14. During which PCR stage are the two strands of double-stranded DNA separated by heat?
Extension
Annealing
Denaturation
Ligation
15. An unknown sample shows no target signal and its internal amplification control also fails, while the positive and no-template controls behave correctly. What should be suspected?
The target is definitively absent from the sample
The entire run is contaminated with the target amplicon
The sample may contain inhibitors or have another sample-specific amplification problem
The positive control proves that every sample contains the target
16. For a typical PCR assay, how should the two primers be oriented relative to the target region?
They should bind entirely outside the template molecule
Their 5′ ends should be joined to each other before cycling
Both primers should bind the same template strand in the same direction
Their extendable 3′ ends should point toward the region to be copied