OBJECTIVES: Within the laboratory protocols, used for the study of BCR-ABL resistance mutations in chronic myeloid leukemia patients treated with Imatinib, direct sequencing remains the reference method. Since the incidence of patients with a mutation-related loss of response is not very high, it is very useful in the routine laboratory to perform a fast pre-screening method. DESIGN AND METHODS: With this in mind, we have designed a new technique, based on a single Real-Time FRET-based PCR, followed by a study of melting peaks. This new tool, developed in a LightCycler 2.0, combines four different fluorescence channels for the simultaneous detection, in a single close tube, of critical mutations within the ABL kinase domain. RESULTS: Assay evaluation performed on 33 samples, previously genotyped by sequentiation, resulted in full concordance of results. CONCLUSIONS: This new methodology detects in a few steps the presence of critical mutations associated to Imatinib resistance.
Martinez-Serra J., Gutiérrez A., Marcús T.F., Soverini S., Amat J.C., Navarro-Palou M., et al. (2012). Four-channel asymmetric Real-Time PCR hybridization probe assay: a rapid pre-screening method for critical BCR-ABL kinase domain mutations. CLINICAL BIOCHEMISTRY, 45, 345-351 [10.1016/j.clinbiochem.2011.12.026].
Four-channel asymmetric Real-Time PCR hybridization probe assay: a rapid pre-screening method for critical BCR-ABL kinase domain mutations.
SOVERINI, SIMONA;
2012
Abstract
OBJECTIVES: Within the laboratory protocols, used for the study of BCR-ABL resistance mutations in chronic myeloid leukemia patients treated with Imatinib, direct sequencing remains the reference method. Since the incidence of patients with a mutation-related loss of response is not very high, it is very useful in the routine laboratory to perform a fast pre-screening method. DESIGN AND METHODS: With this in mind, we have designed a new technique, based on a single Real-Time FRET-based PCR, followed by a study of melting peaks. This new tool, developed in a LightCycler 2.0, combines four different fluorescence channels for the simultaneous detection, in a single close tube, of critical mutations within the ABL kinase domain. RESULTS: Assay evaluation performed on 33 samples, previously genotyped by sequentiation, resulted in full concordance of results. CONCLUSIONS: This new methodology detects in a few steps the presence of critical mutations associated to Imatinib resistance.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
