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Clin Biochem. 2012 Mar;45(4-5):345-51. doi: 10.1016/j.clinbiochem.2011.12.026. Epub 2012 Jan 12.

Four-channel asymmetric Real-Time PCR hybridization probe assay: a rapid pre-screening method for critical BCR-ABL kinase domain mutations.

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  • 1Department of Hematology, University Hospital of Son Espases, Carretera Valldemossa no. 79, Palma de Mallorca, Spain. jorgej.martinez@ssib.es

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.

Copyright © 2012 Elsevier Inc. All rights reserved.

PMID:
22266405
[PubMed - indexed for MEDLINE]
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