Mixed Deck — All PCR Topics Flashcards

Which tissue fixative is least suitable for subsequent PCR analysis?

Answer: Bouin's solution

Bouin's contains picric acid, which severely degrades nucleic acids.

In a SYBR Green assay, the no-template control (NTC) amplifies at Ct 37 and its melt peak is well below the target Tm. What is the best interpretation?

Answer: Primer-dimer formation in the NTC

Late NTC amplification with a low-Tm melt peak is typical of primer dimers, not contaminating target.

A DNA extract has A260/A280 of 1.85 but A260/A230 of 0.9. What is the most likely contaminant?

Answer: Chaotropic salts such as guanidine, or carbohydrates

A low A260/A230 ratio indicates contaminants absorbing at 230 nm, such as guanidine salts, phenol, or carbohydrates.

Which method best verifies temperature uniformity across a thermal cycler block?

Answer: A multi-probe temperature verification system placed in multiple wells

Multi-probe systems measure several well positions at the same time, which shows differences across the block.

What is a common specimen-handling cause of false-positive PCR results?

Answer: Cross-contamination from aerosols or amplicon carryover

Tiny amounts of contaminating nucleic acid can be amplified, producing false positives.

Why is cycle number important in PCR?

Answer: It controls DNA yield and fidelity

The number of PCR cycles directly influences both the quantity and quality of the amplified DNA. Too few cycles result in insufficient product yield, while too many cycles can lead to a plateau effect, accumulation of errors, and the formation of non-specific products due to reagent depletion or increased chance of mispriming. Therefore, optimizing cycle number is crucial for achieving adequate DNA yield while maintaining the fidelity of the amplified product.

In SI-compliant scientific writing, how should a denaturation temperature be written?

Answer: 95 °C

SI style puts a space between the number and the unit symbol, including °C.

Why should environmental swab monitoring include a negative swab control (an unused swab processed alongside samples)?

Answer: To confirm the swabs and extraction reagents are not themselves contaminated

An unused swab control shows whether contamination comes from the sampling materials rather than the surface.

Which element helps avoid non-specific amplification?

Answer: Hot-start polymerase

Hot-start polymerases are chemically modified or antibody-bound enzymes that are inactive at room temperature and only become active after an initial high-temperature denaturation step. This prevents non-specific primer binding and amplification that can occur at lower temperatures before the main PCR cycling begins. By minimizing activity during setup, hot-start polymerases significantly reduce the formation of non-specific products like primer dimers.

What is the purpose of using proteinase K in DNA extraction?

Answer: Degrades proteins in the sample

Proteinase K is an enzyme used in DNA extraction to degrade proteins present in the sample. Its primary purpose is to break down cellular proteins, including nucleases that could degrade DNA, and histones that are tightly bound to DNA. This enzymatic digestion helps to purify the DNA by removing protein contaminants, ensuring a cleaner and more stable DNA sample.

Which control best detects PCR inhibition in an individual environmental sample extract?

Answer: An internal amplification control (IAC) spiked into each reaction

An IAC amplified in the sample's own reaction shows whether that matrix suppresses amplification.

What role does documentation play in scientific writing & communication within Polymerase Chain Reaction?

Answer: It ensures continuity, accountability, and serves as a reference for all parties

Proper documentation ensures continuity of care or service, establishes accountability, and provides a reliable reference for all involved parties.

During pipette calibration, the coefficient of variation (CV) of repeated dispenses measures:

Answer: Precision (random error)

CV reflects how closely repeated dispenses agree, which is precision or random error.

How does ongoing professional development support regulatory standards & compliance in Polymerase Chain Reaction?

Answer: It keeps professionals informed of evolving standards and best practices

Ongoing professional development ensures that practitioners stay current with evolving regulations, standards, and best practices in their field.

Your PCR products run as a smear on an agarose gel. Which change is most likely to help?

Answer: Lower the amount of template or the number of cycles

Smears often come from too much template or too many cycles, which cause nonspecific products and over-amplification.

What is the main purpose of aerosol-resistant (filter) pipette tips in PCR setup?

Answer: To block aerosols from contaminating the pipette barrel and subsequent samples

Filter barriers stop aerosolized DNA from entering the pipette shaft and cross-contaminating later reactions.

What documentation is ESSENTIAL for instrumentation & calibration management in Polymerase Chain Reaction?

Answer: Maintenance logs, calibration records, and incident reports

Maintenance logs, calibration records, and incident reports provide a complete history that supports safety, compliance, and lifecycle management.

What is a key hazard when using liquid nitrogen or large amounts of dry ice in a poorly ventilated room?

Answer: Oxygen displacement leading to asphyxiation

Evaporating nitrogen or sublimating CO2 can displace oxygen in confined spaces.

What is the recommended long-term storage condition for purified RNA?

Answer: -80°C, ideally aliquoted in RNase-free buffer or water

Storing RNA at -80°C in aliquots minimizes degradation from RNases and repeated freeze-thaw cycles.

What is the MOST important factor in instrumentation & calibration selection for Polymerase Chain Reaction?

Answer: Suitability for the intended purpose and compliance with applicable standards

Equipment must be suitable for its intended purpose and comply with applicable standards to ensure safety and effectiveness.