Non-radioactive detection of the rearranged BCR/ABL sequences amplified by polymerase chain reaction.
The polymerase chain reaction (PCR) is a powerful technique for the detection of the bcr/abl rearrangement in chronic myelogenous leukemia (CML). It allows the exponential amplification of the rearranged region, thus facilitating its detection. The specificity of the bcr/abl cDNA sequence amplified by PCR is most commonly verified by hybridization to a 32P-labeled probe. In this paper, an assay for the colorimetric detection of the amplified bcr/abl fragment is described, which offers several advantages over the use of radioactive probes. We adapted the PCR for synthesis and simultaneous labeling of DNA fragments with a non-radioactive steroid compound called digoxigenin. This labeling procedure was used to generate a digoxigenin-labeled internal probe for the chimeric bcr/abl mRNA. The assay described is based on the hybridization of the amplified bcr/abl sequence to the non-radioactively labeled probe and on the subsequent detection by an enzyme-linked immunoassay and enzyme-catalyzed color reaction. Using this protocol, we investigated 20 patients with CML along with six healthy individuals and two cell lines derived from patients with CML for the presence of the bcr/abl rearrangement. It is shown that the assay is both highly sensitive and specific and that it is readily applicable to the routine diagnosis of CML. In addition, the assay could be adapted to a number of clinical diagnostic uses.
Lion
T
T
Children's Cancer Research Institute, St Anna Kinderspital, Wien, Austria.
Prischl
F
F
Haas
O A
OA
Pont
J
J
Schwarzmeier
J
J
D003124:Colorimetry; D004076:Digoxigenin; D016044:Fusion Proteins, bcr-abl / Q000235:genetics*; D015321:Gene Rearrangement; D006801:Humans; D015151:Immunoblotting; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000175:diagnosis; D016133:Polymerase Chain Reaction
D016044:Fusion Proteins, bcr-abl; D004076:Digoxigenin