While the methods above are designed to reduce PD formation, another approach aims to minimize signal generated from PDs in quantitative PCR. This approach is useful as long as there are few PDs formed and their inhibitory effect on product accumulation is minor.
Four steps PCR: used when working with nonspecific dyes, such as SYBR Green I. It is based on the different length, and hence, different melting temperature of the PDs and the target sequence. In this method the signal is acquired below the melting temperature of the target sequence, but above the melting temperature of the PDs.
Sequence-specific probes: TaqMan and molecular beacon probes generate signal only in the presence of their target (complementary) sequence, and this enhanced specificity precludes signal acquisition (but not possible inhibitory effects on product accumulation) from PDs.