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Detection of novel NF2 mutations by an RNA mismatch cleavage method.

Faudoa R., Xue Z., Lee F., Baser M.E., Hung G.

Mutations in the neurofibromatosis type 2 gene (NF2) cause benign nervous system tumors. Common methods for detecting NF2 mutations (such as single stranded conformational polymorphism analysis and denaturing gradient gel electrophoresis) are laborious and time-consuming. We adapted and improved a commercial assay, the Non-Isotopic RNase Cleavage Assay (NIRCAtrade mark, Ambion, Austin, TX) for rapid, non-isotopic, high-sensitivity screening for NF2 mutations in tumors. We improved the assay by: 1) extending the typical NIRCAtrade mark template size of < 500 bp to 1.3 kb without decreasing detection efficiency; 2) modifying the transcription step of the original protocol so that transcription of PCR products was increased by up to 50%; 3) optimizing the combination of cleavage enzymes and reaction time. With these modifications, mutations were found in 15 of 20 patients (75%) using NIRCAtrade mark. Seven of the point mutations detected (two nonsense, two missense, and three splice-site) are novel. All mutations were confirmed by direct sequencing and no mutations were found using direct sequencing in patients that were negative by NIRCAtrade mark. The 75% NF2 mutation detection rate using this design is similar to detection rates in tumors using other mutation detection methods.

Hum. Mutat. 15:474-478(2000) [PubMed] [Europe PMC]

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