Mutations in exon 12 of the nucleophosmin gene (NPM1) that cause the encoded protein to abnormally relocate to the cytoplasm are found at diagnosis in about 50% of karyotypically normal acute myeloid leukemias and are associated with a more favorable outcome. We have devised a PCR-based assay for NPM1 exon 12 mutations using differential melting of an oligo probe labeled with a fluorescent dye. The nucleobase quenching (NBQ) phenomenon was used to detect probe hybridization, and an oligonucleotide containing locked nucleic acid (LNA) nucleotides was used as a PCR clamp to suppress amplification of the normal sequence and enhance the analytical sensitivity of the assay. After the NBQ assay, the specimens with a mutation were removed from the capillary and sequenced to identify the mutation. The use of the LNA clamp facilitates interpretation of the mutant sequence because of the lower intensity of the overlapping normal sequence. Analysis of a series of 70 patient specimens revealed 17 positive for an NPM1 mutation and 53 negatives. All of the NBQ results (positives and negatives) were confirmed with other methods. The analytical sensitivity of the NBQ assay is variable depending on the concentration of the PCR clamp and other parameters. Using a 100 nmol/L concentration of the LNA clamp, NPM1 mutations were detectable in a 10-fold excess of wild-type DNA. This assay may be valuable for screening disease specimens.