METHOD FOR QUANTITATIVE DETERMINATION OF G250E MUTATION OF BCR-ABL KINASE DOMAIN FOR PATIENTS WITH CHRONIC MYELOID LEUKOSIS RESISTANT TO THYROSINKINASE INHIBITORS THERAPY Russian patent published in 2017 - IPC C12N15/00 

FIELD: biotechnology.

SUBSTANCE: method for identification of G250E mutation of BCR-ABL kinase domain for chronic myeloid leukemia (CML) patients. The claimed method includes real-time polymerase chain reaction using cDNA obtained by the method of reverse transcription from RNA isolated from blood samples of patients with chronic myeloid leukemia resistant to tyrosine kinases inhibitors of the first generation. A system consisting of a forward primer SEQ ID NO: 1 and a reverse primer specific for mutation SEQ ID NO: 2 is used as primers. To quantify G250E mutation, a specific probe, SEQ ID NO: 3, is used. Number of BCR-ABL^G250E copies in samples are determined from the calibration curve. To determine the total BCR-ABL expression, a system is used consisting of a forward primer, SEQ ID NO: 4, a reverse primer, SEQ ID NO: 5 and a specific probe SEQ ID NO: 6. To determine the control ABL gene expression, a system is used consisting of a forward primer, SEQ ID NO: 7, a reverse primer, SEQ ID NO: 8, and a specific probe of SEQ ID NO: 9. BCR-ABL^G250E expression is calculated relative to the total expression of BCR-ABL and control ABL gene by the formula: (Q BCR-ABL^G250E av./Q ABL av.)/(Q BCR-ABL av./Q ABL av.)*100%.

EFFECT: method allows to increase sensitivity and specificity of the mutant clone amount and reduce research time.

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