Optimized non-radioactive protein truncation test for mutation analysis of the adenomatous polyposis coli (APC) gene.
Germline mutations in the adenomatous polyposis coli gene cause familial 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 the protein truncation test is ideally suited for screening of mutations in these genes, starting from simple blood samples. Gene segments of interest are amplified from genomic DNA or mRNA, thereby incorporating a T7 promoter at the 5'-end. After in vitro transcription and translation of the PCR products, the resulting protein is analysed by gel electrophoresis. Truncated translation products indicate the presence of a stop mutation. We have developed a non-radioactive protein truncation test that uses a biotinylated Lys-t-RNA to label the translation products 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 adenomatous polyposis coli. The adenomatous polyposis coli gene was divided in 5 overlapping segments, and primers were optimized to produce distinct bands with very low background in the protein truncation test. The assay was tested on 20 familial adenomatous polyposis patient samples, where 18 mutations were found, demonstrating the efficiency of this method.
Kirchgesser
M
M
Boehringer Mannheim GmbH, Penzberg, Germany. Michael.Kirchgesser@bmg.boehringer-mannheim.com
Albers
A
A
Vossen
R
R
den Dunnen
J
J
van Ommen
G J
GJ
Gebert
J
J
Dupont
C
C
Herfarth
C
C
von Knebel-Doeberitz
M
M
Schmitz-Agheguian
G
G
D011125:Adenomatous Polyposis Coli / Q000235:genetics*; D017491:Genes, APC; D018095:Germ-Line Mutation; D006801:Humans; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011506:Proteins / Q000737:chemistry / Q000235:genetics*; D014158:Transcription, Genetic
D011506:Proteins