TaqMan reverse transcriptase-polymerase chain reaction coupled with capillary electrophoresis for quantification and...

Real-time TaqMan polymerase chain reaction (PCR) assays allow quantification of the initial amount of target in a specimen, specifically, and reproducibly. The major limitation of TaqMan PCR assays is that they do not detect the size of the amplified target sequence. TaqMan PCR coupled with capillary electrophoresis is an alternative approach that can be used to circumvent this limitation. In this chapter, the utility of this approach in the identification and quantification of bcr-abl fusion transcripts produced as a result of t(9;22)(q34;q11) in chronic myelogenous leukemia is described. In this assay, abl primer labeled at its 5'-end with the fluorescent dye NED (Applied Biosystems [ABI], Foster City, CA) is incorporated into the bcr-abl fusion product during the real-time PCR. The incorporated NED fluorescent dye is then used subsequently to identify the specific fusion transcript present in a given specimen by high-resolution capillary electrophoresis and GeneScan (ABI) analysis. Knowledge of the type of fusion transcript present in a specimen is useful to rule out false-positive results and to compare clones before and after therapy.