Rapid identification of RT-PCR clones containing translation-terminating mutations

The technique of in vitro transcription/translation (IVTT) has become an important method of detecting mutations that result in a prematurely terminated protein. Subsequent characterization of the mutations by cloning and sequencing the RT-PCR products, however, is often difficult and time consuming. This is due in large part to the altered metabolism to which transcripts containing translation terminating mutations are subject. Recent data has shown that mRNAs with nonsense or frame shift mutations are often selectively degraded, so that mutation-bearing transcripts are significantly less abundant than wild-type transcripts and, after cloning, mutant clones are correspondingly scarce. We have developed a reliable method of identifying the cDNA clones containing translation terminating mutations by a 'second round' of IVTT, Clones are subjected to PCR and IVTT using similar conditions as in the initial IVTT reaction and are identified unequivocally as either wild-type or mutant prior to sequencing. Wasteful 'blind' sequencing is thus avoided as well as possible misidentification of tag polymerase errors as the mutation of interest.