|dc.description.abstract||Background: The main cause to chronic myeloid leukemia (CML) is a translocation between chromosome 9 and 22, resulting in the Philadelphia chromosome, containing a BCR-ABL fusion-gene that encodes a constitutively active tyrosine kinase. Imatinib, a selective inhibitor of the tyrosine kinase, is the first-line treatment for CML-patients. However, drug-resistance is an increasing problem, and the most important reasons for this are point mutations within the BCR-ABL kinase domain. More than 40 such mutations are described, and because the mutations can have a great impact on clinical decision-making, early detection is important. With new tyrosine kinases developing, the resistance problem seems to be diminished, as some of the new drugs are less prone to resistance. Only the important T3151-mutation seems to remain a problem, which is why this particular mutation should be in focus for developing a more sensitive detection method.
Materials and methods: Plasmids and patient-samples containing wild-type and mutated BCR-ABL sequences were used in an effort to design a sensitive and specific, biased assay for detection of the T3151-mutation in BCR-ABL alleles. By comparing a single-step versus a nested approach, the amplification of long versus short templates, TaqMan versus SYBR Green Master Mixes, and ASO versus LNA and ARMS-primers, a patient-test for detection of the T3151-mutation was developed.
Results: A nested PCR approach using SYBR Green Master Mix and BCR-ABL-specific primer pair with a LNA-nucleotide incorporated in the mutation-specific reverse primer provided the greatest improvements in sensitivity and specificity for detection of the T3151-mutation in samples from CML-patients.||en