OPTIMIZED NONRADIOACTIVE PROTEIN TRUNCATION TEST FOR MUTATION ANALYSIS OF THE ADENOMATOUS POLYPOSIS-COLI (APC) GENE

Germline mutations in the adenomatous polyposis coli gene cause famili al adenomatous polyposis, a colon cancer predisposition syndrome. More than 95% of the identified mutations result in the generation of stop codons or reading frame shifts and encode a truncated gene product, a mutation profile also found in other tumor predisposition genes such as the breast cancer or the hereditary non-polyposis coli. Therefore t he protein truncation test is ideally suited for screening of mutation s in these genes, starting from simple blood samples. Gene segments of interest are amplified from genomic DNA or mRNA, thereby incorporatin g a T7 promoter at the 5'-end. After in vitro transcription and transl ation of the PCR products, the resulting protein is analysed by gel el ectrophoresis. Truncated translation products indicate the presence of a stop mutation. We have developed a non-radioactive protein truncati on test that uses a biotinylated Lys-t-RNA to label the translation pr oducts and allows a chemiluminescent detection instead of the standard radioactive method. This generic protein truncation test kit was then used to develop a parameter-specific protein truncation test for aden omatous polyposis coli. The adenomatous polyposis coli gene was divide d in 5 overlapping segments, and primers were optimized to produce dis tinct bands with very low background in the protein truncation test. T he assay was tested on 20 familial adenomatous polyposis patient sampl es, where 18 mutations were found, demonstrating the efficiency of thi s method.