Real-time PCR quantification strategy for IgH tumor-related sequences and TaqMan probe sequences.

(A) Quantitative PCR is performed with appropriate V_Hconsensus primer, patient tumor-specific (ASO) primer, and V_H gene family-specific consensus TaqMan probe. Antisense probes are labeled at the 5′ end with reporter dye (R) 6-carboxy fluorescein (FAM) and at the 3′ end with quencher activity (Q) 6-carboxy-tetramethyl rhodamine (TAMRA). (B) Sequences of V_H gene family-specific consensus TaqMan probes. (C) Representative amplification plot showing increasing fluorescence (ΔRn) detected through the final 37 cycles of a 40-cycle PCR amplification reaction on a 10-fold serial dilution of a plasmid-encoded tumor-related V_H5 IgH sequence with the V_H5 consensus probe (IgH copy numbers ranging from 10⁶ to 0). Ct represents the threshold cycle at which fluorescence is first detected above background.