BCR-ABL1 kinase structural domain mutation screening method

Abstract

The invention discloses a BCR-ABL1 kinase structural domain mutation screening method. The method comprises the following steps: separating mononuclear cells from a sample, extracting RNA, carrying out reverse transcription on the extracted RNA to obtain cDNA, carrying out PCR and purification to obtain an amplification product comprising a BCR-ABL1 kinase structural domain, carrying out sequencing library construction on an amplification product, sequencing the sequencing library on a PGM sequencing platform, determining mutation sites and mutation allele frequencies thereof according to a sequencing result, and judging whether mutation sites and mutation modes for TKI drug resistance appear or not according to the mutation allele frequencies of the mutation sites. The screening method provided by the invention can accurately obtain mutation sites and variant allele frequencies thereof in a sample sequence, and determine low-level mutation (less than 20%), composite mutation and polyclonal mutation, besides, the screening method is combined with the characteristics of nested PCR, and specific primers are matched for use, so that the screening method disclosed by the invention hasrelatively high sensitivity and specificity and relatively high screening sensitivity.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for screening mutations in the kinase domain of BCR-ABL1. Background technique [0002] BCR-ABL1 kinase domain (KD) mutations in BCR-ABL1 fusion-positive leukemia patients are a common mechanism of resistance to tyrosine kinase inhibitors (TKIs). In almost all studies, mutations in BCR-ABL1 KD were detectable in 50%-80% of patients when disease relapsed. These mutations suggested high IC50 values ​​in biochemical and cellular assays. The most common imatinib-resistant mutation site in BCR-ABL1-positive leukemia patients is T315I. Except for ponatinib or monoclonal antibodies, other second-generation TKIs (2G-TKIs) are ineffective against T315I. Mutation-positive patients exhibit extremely high genetic instability, which may contribute to subpopulation of cells positive for the same ("compound" mutations, compound mutations) or different ("polyclonal" mutations, polyclonal ...

AI Technical Summary

Problems solved by technology

However, while Sanger sequencing (SS) testing is the gold standard for BCR-ABL1 KD mutation screening, the nature of this method makes it unsuitable for identifying low-level variants (<20% VAF) and often cannot clearly distinguish Multiple mutations occurring in the same clone (composite mutations) or in separate clones (polyclonal mutations)

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