Detection of NF1 Mutations Utilizing the Protein Truncation Test (PTT)

The protein truncation test (PTT) (1 –3 ), also known as the in vitro coupled transcription/ translation synthesis assay, was designed as a tool to detect mutations that lead to premature translational termination. It was originally developed to screen for mutations in the dystrophin (DMD) gene causing Duchenne/Becker muscular dystrophies. The ability of the PTT to detect mutations at the protein level offers various advantages over other screening methods such as single-strand conformational polymorphism analysis (SSCP), heteroduplex analysis (HA), and denaturing gradient gel electrophoresis (DGGE) that reveal polymorphisms and rare variants that may not be disease causing. The PTT has been applied to the mutation screening of a number of large and complex genes including BRCA1 (4 ), BRCA2 (5 ), ATM (6 ), TSC2 (7 ) and NF2 (8 ). For BRCA1 and BRCA2 , more than 3kb of coding sequence can be screened in a single PCR reaction combined with coupled in vitro transcription/translation reaction and run on a single lane on an sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel (9 ).