The Protein Truncation Test

Abstract

The protein truncation test (PTT) (1), occasionally referred to as the in vitro synthesized-protein (IVSP) assay (2), is a method for screening the coding region of a gene for mutations that result in the premature termination of mRNA translation. The techniques involved in performing PTT are relatively straightforward and begin with the isolation of genomic DNA or RNA. The polymerase chain reaction (PCR) is used to amplify a DNA template, usually of 1–3 kb in size, that is tested in an in vitro transcription and translation assay. Truncated proteins are identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography or fluorography. A single, large exon of a gene can be amplified directly from genomic DNA in several overlapping fragments. The complete coding sequence of a large gene, with many small exons, can be amplified in several overlapping fragments by reverse-transcription PCR (RT-PCR) starting from RNA. Amplifying the gene in several segments that overlap each other by 300–500 bp increases the sensitivity of PTT. A truncating mutation located toward the 3′ end of one segment will also occur near the 5′ end of the next overlapping segment, thus increasing the likelihood of identifying a truncated protein. A specially modified forward PCR primer is required for PTT. In addition to the in-frame gene-specific primer sequence, an extension at the 5′ end includes the T7 promoter sequence for RNA transcription by T7 RNA polymerase, the eukaryotic consensus sequence for the initiation of protein translation, and the ATG protein translation start site.