INTRODUCTION OF A MYC REPORTER TAG TO IMPROVE THE QUALITY OF MUTATIONDETECTION USING THE PROTEIN TRUNCATION TEST

The protein truncation test is a useful method for identifying framesh ift and nonsense mutations, The interpretation of real results is ofte n made difficult by the presence of internally translated protein prod ucts and other nonspecific bands. To overcome these problems, the tech nique has been modified to include a myc reporter tag in the 5' primer used to amplify the genomic region of interest. The myc tag is recogn ised by a monoclonal antibody, which can be used to manipulate the pro ducts of the protein truncation test using immunoprecipitation techniq ues, The presence of the myc tag also introduces a mechanism whereby t he use of radioisotopes can be avoided, relying instead on the identif ication of newly synthesised proteins using Western blot technology. T he myc-tag modification can be integrated into all protein truncation tests, regardless of the gene being examined, with only one monoclonal antibody required for the immunoprecipitation purification and Wester n blotting. (C) 1997 Wiley-Liss, Inc.