Fluorescence melting curve analysis for the detection of the bcl-1/JH translocation in mantle cell lymphoma

PCR amplification and product analysis for the detection of chromosomal tra nslocations such as bcl-1/JH have traditionally been performed as a two-ste p process with separate amplification and product detection. PCR product de tection has generally entailed gel electrophoresis, hybridization, or seque ncing for confirmation of assay specificity. By using a microvolume fluorim eter integrated with a thermal cycler and the PCR compatible double-strande d DNA (dsDNA) binding dye SYBR Green I, we simultaneously amplified and det ected bcl-1/JH translocation products by using rapid cycle PCR and fluoresc ence melting curve analysis. We analyzed DNA from 25 cases of lymphoprolife rative disorders comprising 12 previously documented bcl-1/JH-positive mant le cell lymphomas, and 13 reactive lymphadenopathies. The samples were code d and analyzed in a blind manner for the presence of bcl-1/JH translocation s by fluorescence melting curve analysis. The results of fluorescence analy sis were compared with those of conventional PCR and gel electrophoresis. A ll of the 12 cases (100%) previously determined to be bcl-1/JH positive by conventional PCR analysis showed a characteristic sharp decrease in fluores cence at about 86 degrees C by melting curve analysis. For easier visualiza tion of melting temperatures (Tm), fluorescence melting peaks were obtained by plotting the negative derivative of fluorescence over temperature (-dF/ dT) versus temperature (T). Dilutional assays revealed that fluorescence me lting curve analysis was more sensitive than conventional PCR and agarose g el electrophoresis with ultraviolet transillumination by as much as 40-fold . Our results indicate that nucleic acid amplification integrated with fluo rescence melting curve analysis is a simple, reliable, sensitive, and rapid method for the detection of bcl-1/JH translocations. The feasibility of sp ecific PCR product detection without electrophoresis or expensive fluoresce ntly labeled probes makes this methodology attractive for studies in molecu lar pathology.