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Technical Advance |
From the Departments of Pathology*
and
Hematology,
The University of Texas M.D.
Anderson Cancer Center, Houston, Texas
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.
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