Quantitaion Of BCR-ABL Fusion Gene MRNA By Competitive RT-PCR And Capillary Electrophoresis

Philadelphia(ph) chromosome or the bcr-abl fusion gene is the hallmark of chronic myelogenous leukemia and serves as a prognostic marker during its treatment. The most commonly used assays to detect this fusion gene are qualitative, but qualitative technique is considered to have little predictability in individual patients, and is not adapted to the follow-up of redidual disease in patients treated with BMT or IFN. We have developed a competitive RT- PCR assay to quantitate the fusion gene mRNA transctipt and utilized capillary electrophoresis (CE) as a direct and accurate detection of the amplified product. After studying the restriction endonuclease sites, we synthesized a new downstream primer which was I 6bp-longer than the former one but 4 nts of the EaeI site were deleted in it, the 4bp-shorter competitive referance were obtained by mutagenesis PCR amplification. The competitor cDNAs. share the same primer sequence as the cellular eDNA and then were cloned into a pGEM-T vector. Thus we constructed two plasmids named J13 196 and JB 121, their validity was verified with restriction endonuclease and the co-PCR products of the original and competitive cDNA can be easily separeted by CE. Finally the competitive eDNA were transcripted into cRNA in vitro as the internal standard in the RT-PCR assay. Cellular total RNA from CML cells was done RT-PCR procedure together with its corresponding competitive RNA which were serial diluted. The RT-PCR products were analyzed by CE, and amounts of both the target and competitive products were assessed. According to the theory of competitive PCR, the log of reaction product ratios has a simple linear relationship to initial copy number of terget and competitor templates. So the results were analyzed by SPSS statistics package of software, from which the exact numbers of transcript were obtained. Our results confirm that 1) mutagenesis by PCR is a simple and valid method to construct competitive templates; 2) CE analysis offers greater resolution and enhanced sensitivity for detection and quantification of PCR products; 3) the competitive RT-PCR assay for BCR-ABL mRNA was precise and reproducible and is therefore of great clinicle relevance.