PCR Primer Design & Optimization 2

Question 1

What is the recommended primer length range for standard PCR applications?

Accepted Answer

15–30 nucleotides

Answer

5–10 nucleotides

Answer

35–50 nucleotides

Answer

55–70 nucleotides

Question 2

Which in silico tool is most commonly used to check primer specificity against a genome database?

Accepted Answer

BLAST (Basic Local Alignment Search Tool)

Answer

ClustalW

Answer

Phred/Phrap

Answer

GATK HaplotypeCaller

Question 3

A hairpin structure in a primer is problematic primarily because it:

Accepted Answer

Sequesters the 3' end, preventing template binding and extension

Answer

Increases the primer's Tm

Answer

Causes the primer to degrade at room temperature

Answer

Increases the rate of nucleotide misincorporation

Question 4

When designing primers for a region with high GC content, which modification can improve amplification efficiency?

Accepted Answer

Adding DMSO (dimethyl sulfoxide) to the PCR reaction

Answer

Increasing the extension time and adding a GC-rich enhancer

Answer

Reducing the primer concentration to 50 nM

Answer

Using a lower-fidelity polymerase

Question 5

Primer concentration in a standard PCR reaction is typically in which range?

Accepted Answer

0.1–0.5 µM

Answer

0.01–0.05 µM

Answer

1–5 µM

Answer

10–50 µM

Question 6

Which characteristic of a primer pair is essential for multiplexed PCR to avoid inter-primer interference?

Accepted Answer

All primers must have compatible Tm values and no cross-complementarity

Answer

Both primers should have identical sequences

Answer

All primers should be the same length

Answer

Each primer pair should target overlapping amplicons

Question 7

To amplify a cDNA template produced by reverse transcription, what additional consideration must primer design account for?

Accepted Answer

Primers must span an exon-exon junction to ensure genomic DNA contamination is detectable

Answer

Primers must be located within intron sequences

Answer

Primer Tm must be reduced by 15°C to accommodate RNA

Answer

Primers must target the poly-A tail exclusively