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Clinical Pathology

Real-Time 5′ → 3′ Exonuclease-Based PCR Assay for Detection of the t(11;14)(q13;q32)

Abstract

We describe the usefulness of a real-time polymerase chain reaction (PCR) assay for detection of the t(ll;14)(q13;q32), most commonly present in mantle cell lymphoma (MCL). This assay is based on the 5′ → 3′ exonuclease activity of Taq polymerase, which cleaves an internal probe labeled with a reporter dye at its 5’ end and a quencher dye at its 3′ end during PCR. The realtime t(ll;14) PCR assay was established using DNA from a case of MCL with the t(ll;14), amplifiable using conventional PCR and primers specific for the major translocation cluster (MTC) region of the bcl-7 locus and the immunoglobulin heavy chain joining region gene (JH). The specificity was determined by analyzing DNA from 82 cases: 50 MCL, 27 other types of non-Hodgkin lymphoma (NHL), and 5 reactive lymphoid proliferations. The real-time t(ll;14) PCR results were correlated with data obtained by a conventional PCR assay. By using the real-time assay, bcl-7 MTC/JH DNA fusion sequences were detected in 25 of 50 MCLs but not in other NHLs or reactive lymphoid proliferations. Concordance between real-time and conventional PCR methods for MCL was 96% and for all samples was 98%. The results demonstrate that this real-time PCR method to detect bcl-7 MTC/JH DNA fusion sequences is specific and reliable. In addition, the results are available immediately following amplification, without standard post-PCR manipulations.

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