Identification Of T315I Point Mutation Of Bcr/abl Fusion Gene By Mutant Enriched AS-PCR Assay Method And Clinical Application

Objective:Chronic myelogenous leukemia (CML) is a malignant clonal disease derivingfrom the hematopoietic stem cell.90%of patients can be detected characteristic Phchromosome and molecular markers bcr/abl fusion gene, which is located in9q34ofabl oncogene break and translocation to the breaking point clustering22q11regionformed bcr fusion bcr/abl fusion gene. With a wide useful range of molecular targetedtherapy, bcr/abl fusion gene mutation plays an important role throughout thetreatment and prognosis and it is the main cause of poor treatment. The study foundthat T315I mutation is the most difficult to overcome mutation of CML resistancemutations. It is reports that the incidence rate of T315I mutation is about15%inpatients with CML, given the current domestic research on bcr/ablT315Imutation rarelyreported, therefore, this study aimed establish to a viable enzyme enrichment allelespecific PCR (enzyme enrichment AS-PCR) detection of T315I mutation detectiontechnology while providing theoretical basis for CML molecular targeted therapy andfollow-up study.Methods:1. Two pairs of primers were designed according to the gene sequence of abl,and mutated base was introduced into the primer. The genomic DNA of healthyhuman peripheral blood served as template. The wild type exon6fragment and theT315I mutant of abl were amplified by PCR. Then the PCR products were insertedinto pSG5M-flag plasmid and the recombinant plasmids were identified by enzymedigestion and DNA sequencing.2. Establishment of enzyme enrichment AS-PCR method. A pair of universalprimers for wild-type and T315I mutant gene sequence were designed. The genomicDNA of wild-type and T315I-type plasmid served as template, fragment which wasdigested was amplified by PCR. The AS-PCR for T315I gene mutation detection wasestablished based on a pair of specific primers used digested fragments as templates. To optimize the reaction system and conditions. The sensitivity and specificity wasanalyzed.Results:1. We constructed the wild-type pSG5M-flag-abl(wild) and mutant-typepSG5M-flag-abl(T315I) recombinant plasmid standards successfully. Restrictionanalysis and sequencing results were consistent with the expected.2. We established the enzyme enrichment AS-PCR method to detect abl geneT315I mutation successfully. The method sensitivity is0.18%, there is not a falsepositive, the specificity of the method is better.Conclusions:1. We constructed wild type pSG5M-flag-abl(wild) and mutant pSG5M-flag-abl(T315I) recombinant plasmids successfully.2. We established the enzyme enrichment AS-PCR method to detect the T315Ipoint mutation of abl gene successfully. To provide a novel method with higherspecificity and sensitivity for the clinical detection of T315I point mutation.