5 '-> 3 ' exonuclease-based real-time PCR methods for detecting the t(14;18) and t(11;14) in non-Hodgkin's lymphomas

Polymerase chain reaction (PCR) assays traditionally require-a number of po st-PCR steps to detect the amplified reaction products. These post-PCR step s include gel electrophoresis and staining with dyes, such as ethidium brom ide, that bind to double stranded DNA. When high levels of sensitivity and specificity are required, additional steps may be done including Southern b lot transfer of products to a membrane and hybridization with specific prob es tagged with signaling systems (e.g., radioactive phosphorus or chemilumi nescent compounds). Real-time PCR assays obviate the need for relatively la bor intensive post-PCR steps, improving turnaround time and reducing the ri sk of contamination. Many different approaches to realtime BCR have been de veloped. In this review, we describe our experience using 5'-->3' exonuclea se-based real-time PCR assays to assess for the t(14;18)(q32;q21) and t(11; 14)(q13;q32) in non-Hodgkin's lymphomas. We combined these real-time assays with the PRISM(TM) 7700 Sequence Detector (PE Applied Biosystems), which h as both a laser-based detection system and computer software for data analy sis. In our laboratory, these assays are as specific as conventional PCR me thods and more convenient. This approach also facilitates more accurate qua ntification of the reaction products.