CA_5 MmD

What is one of the most crucial factors affecting the success and quality of quantitative real-time PCR (qPCR) analyses?

• The size of the PCR machine

• The design of primers and probes

• The color of the primers

• The type of DNA sample used

What should be avoided or reduced when designing primers and probes for qPCR?

• High GC content

• Formation of primer-dimers

• Long amplicon length

• Formation of hairpins

What is the recommended amplicon length for high efficiency of qPCR?

• 1000 bp

• 400 bp

• 200 bp

• 50 bp

What is the recommended range for the melting temperature (Tm) of qPCR primers?

• 40-45°C

• 55-60°C

• 70-75°C

• 80-85°C

Why should primers and probes with high self-complementarity be avoided?

• To avoid primer-dimers

• To prevent hairpin formation

• To improve amplification efficiency

• To increase specificity

What should be excluded from the 5′ end of the probe to avoid continuous quenching?

• T

• A

• G

• C

What is the purpose of using non-fluorescent quenchers in qPCR assays?

• To increase fluorescence intensity

• To reduce background signal

• To improve primer specificity

• To enhance probe binding

Why should primers and probes not overlap or be complementary to each other in qPCR reactions?

• To prevent primer-dimers

• To increase amplification efficiency

• To reduce specificity

• To avoid hairpin formation

What is the recommended GC percentage range for primers and probe sequences?

• 10-20%

• 70-80%

• 30-40%

• 50-60%

What is the main difference between one-step and two-step RT-qPCR procedures?

• The presence of a quencher

• The separation of transcription and amplification steps

• The number of primers used

• The type of DNA sample used