The exonuclease-based real-time polymerase chain reaction (PCR) exploits 5'→3' exonuclease activity of Taq polymerase and measures PCR product accumulation as the reaction proceeds through a dual-labeled fluorogenic probe. The utility of this exonuclease-based PCR assay as a rapid alternative to conventional PCR for follicular lymphoma-associated t(14;18)(q32; q21) was evaluated in this study. The specificity of the assay for t(14;18) involving bcl-2 and immunoglobulin heavy-chain joining region (JH) genes was assessed by analyzing DNA from 53 patients (38 B-cell non-Hodgkin's lymphomas and 15 nonneoplastic proliferations) and correlating the exonuclease PCR data with conventional PCR results. bcl-2/JH fusion sequences were detected by exonuclease-based PCR in 24 of 25 cases shown to be bcl-2 rearranged by conventional PCR. Fusion sequences were not detected in patients who were negative by conventional PCR. The overall concordance between the two assays was 98% (52 of 53 cases concordant positive or negative). In a serial dilution study using t(14;18)-positive cell line DNA, exonuclease-based PCR detected fusion sequences at DNA concentrations of 5 pg, equivalent to 0.6 to 0.8 genomes per reaction. Thus, this study demonstrated that exonuclease- based PCR for t(14; 18) is both specific and highly sensitive. The elimination of the post-PCR amplicon detection steps and the ability to quantitate the input target DNA sequences make this assay ideal for routine diagnostics and monitoring minimal residual disease.
Luthra, R., McBride, J. A., Cabanillas, F., & Sarris, A. (1998). Novel 5’ exonuclease-based real-time PCR assay for the detection of t(14;18)(q32;q21) in patients with follicular lymphoma. American Journal of Pathology, 153(1), 63–68. https://doi.org/10.1016/S0002-9440(10)65546-0