2024-12-04
qPCR primer design follows basic PCR principles, but varies depending on the quantification method. For the SYBR dye method, key considerations include primer length, G+C content, 3' end base selection, avoidance of dimers and hairpin structures, Tm matching, ΔG control, 5' end modification, product length, and intron spanning. In addition to these principles, the TaqMan probe method also requires considerations such as avoiding G bases at the 5' end, a higher probe annealing temperature than th