40P The detection and quantification of different sequence-variable NPM1 mutations using RNase H-dependent PCR (rhPCR)

Molecular diagnostics for the detection and characterization of genetic variants is based in majority cases on PCR technology. Since primers probes are designed according to nucleotide sequence DNA region specific mutation, technology can only be applied if mutation well known. In where mutated a gene may have variable sequences, different between patients, set each variant necessary, which involves large volume work. The nucleophosmin (NPM1) also included this category more than 24 known 6 common. aim our study was establish qPCR assay molecular kit (a primers) quantification NPM1 mutations regardless its by using RNase H-dependent (rhPCR). Several sets rh-primers that show point mismatches relation some been designated tested. carried removable blocker at 3′ end. Only after removal RNaseH2 enzyme, expandable polymerase. used determine extent presence these could affect H2-endonuclease activity ribose digestion activation. evaluation out preclinical context, synthetic fragments carrying (AML type A, B, D, I, K). After establishing optimal combination primer as reaction conditions, sensitivity limit tested samples with number target copies varied decreasingly: 1000000, 100000, 10000, 1000, 100, 10. It observed nature same detect 10 mutant NPM1. main advantage sequence-variable detected quantified high specificity through single rhPCR reaction. no longer necessary design, synthesize optimize individual mutation. Our demonstrates potential use monitoring patients AML minimum residual disease recurrence risk.