4.8. Primer and probe design
There are numerous primer design software packages around to help design primers and, if appropriate, TaqMan® probes for the amplified regions (Yuryev, 2007). Such software generally recommends using very short amplicons (<100 nucleotides), which shortens the cycling times, avoids incomplete amplicons and saves reagents, avoiding competition even at late cycles. However, longer amplicons (up to 500 base pairs) provide much greater flexibility in designing an internal (e.g., TaqMan®) probe for the target. The probe should as much as possible be devoid of secondary structures (stem-loops) and have a Tm slightly higher than that of the amplification primers, so that it anneals to the denatured target molecules before any primer-driven polymerisation takes place. ‘G’ bases should be avoided at the 5’ end, where the fluorophore usually resides, since they quench fluorescence, even after cleavage (Bustin, 2000).