The major mechanism of imatinib resistance for patients with chronic myeloid leukemia (CML) is clonal expansion of leukemic cells with mutations in the Bcr-Abl fusion tyrosine kinase that reduce the capacity of imatinib to inhibit kinase activity. The early detection of such mutations may allow timely treatment intervention to prevent or overcome resistance. Direct sequencing of the BCR-ABL kinase domain is relatively rapid and allows detection of emerging mutations at a sensitivity of approx 20%. Mutations have been detected over a range of 242 amino acids, which spans the entire kinase domain. For optimal sensitivity, the kinase domain of the abnormal gene should be isolated by reverse-transcription (RT) polymerase chain reaction (PCR) amplification using primers that hybridize to the BCR and ABL genes. The quality of the RNA is assessed by real-time quantitative PCR prior to analysis, and BCR-ABL levels are determined. Only RNA of adequate quality is used to ensure accurate and reproducible mutation analysis. Depending on the level of BCR-ABL transcripts, a one- or two-step PCR is required to amplify the kinase domain. Direct sequencing with dye terminator chemistry is performed using PCR-purified products. The sequence is compared to an ABL kinase domain reference sequence using sequencing analysis software, which aligns the sequences and highlights single or multiple mutations.
|Number of pages||14|
|Journal||Methods in molecular medicine|
|Publication status||Published - 1 Jan 2006|
ASJC Scopus subject areas
- Molecular Medicine