Many of the guidelines for primer design are also applicable to TaqMan® probe design. It is recommended to use Primer Express® software (Applied Biosystems) for TaqMan® assay design.
• The probe melting temperature in general should be ~10°C higher than the forward or reverse primer.
• Do not put G at the 5' end of the probe as this will quench reporter fluorescence.
• In general the GC content should be 35-65%.
• The probe should not self-anneal to form secondary structure and should not be picked from a gene region with high likelihood of secondary structure. Secondary structure formation may reduce hybridization efficiency, leading to reduced assay sensitivity.
• The probe should be selected from gene-specific regions with reasonable sequence complexity to avoid cross-hybridization.
• The probe should be as close to the forward and reverse primers as possible, without overlapping the primer sequences. The amplicon size is usually in the range of 60-150.
Was this article helpful?