qPCR system for the FOXP3 TSDR to detect Treg. qPCR system for the FOXP3 TSDR to detect Treg. A, schematic overview of the FOXP3 locus and the amplification strategy. Two methyl-sensitive amplification primers and methyl-sensitive hybridization probes were designed. Lollipops, targeted CpG sites. The detection dye, which is quenched as long as the probe is intact, is released upon exonuclease digestion during specific strand elongation. B, amplification profiles of the qPCR systems (top and middle). Left, nonmethylation-specific amplification system; right, methylation-specific amplification system. Top, a plasmid corresponding to nonmethylated (nm) FOXP3 TSDR DNA (all Cs in the TSDR are replaced by T) is used in a serial dilution row ranging from 200 million to 20 copies. Middle, the same dilution with a plasmid corresponding to methylated (m) FOXP3 TSDR DNA (all Cs other than in the context of CG are replaced by T). Bottom, standard curves as produced by serial dilutions of plasmid present themselves in a strictly log-linear fashion. Georg Wieczorek et al. Cancer Res 2009;69:599-608 ©2009 by American Association for Cancer Research