Optimized non-radioactive protein truncation test for mutation analysis of the adenomatous polyposis coli (APC) gene

Michael Kirchgesser, Andreas Albers, Rolf Vossen, Johan Den Dunnen, Gert Jan Van Ommen, Johannes Gebert, Cecile Dupont, Christian Herfarth, Magnus von Knebel Doeberitz, Gudrun Schmitz-Agheguian

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)567-570
Number of pages4
JournalClinical Chemistry and Laboratory Medicine
Volume36
Issue number8
DOIs
StatePublished - Oct 23 1998

Fingerprint

APC Genes
Adenomatous Polyposis Coli
Genes
Mutation
Proteins
Reading Frames
Germ-Line Mutation
Terminator Codon
Transcription
Electrophoresis
Colonic Neoplasms
Labels
Tumors
Assays
Screening
Blood
Gels
RNA
Breast Neoplasms
Polymerase Chain Reaction

Keywords

  • Adenomatous polyposis coli
  • Mutation analysis
  • Protein truncation test

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Biochemistry, medical

Cite this

Kirchgesser, M., Albers, A., Vossen, R., Den Dunnen, J., Van Ommen, G. J., Gebert, J., ... Schmitz-Agheguian, G. (1998). Optimized non-radioactive protein truncation test for mutation analysis of the adenomatous polyposis coli (APC) gene. Clinical Chemistry and Laboratory Medicine, 36(8), 567-570. https://doi.org/10.1515/CCLM.1998.097

Optimized non-radioactive protein truncation test for mutation analysis of the adenomatous polyposis coli (APC) gene. / Kirchgesser, Michael; Albers, Andreas; Vossen, Rolf; Den Dunnen, Johan; Van Ommen, Gert Jan; Gebert, Johannes; Dupont, Cecile; Herfarth, Christian; von Knebel Doeberitz, Magnus; Schmitz-Agheguian, Gudrun.

In: Clinical Chemistry and Laboratory Medicine, Vol. 36, No. 8, 23.10.1998, p. 567-570.

Research output: Contribution to journalArticle

Kirchgesser, M, Albers, A, Vossen, R, Den Dunnen, J, Van Ommen, GJ, Gebert, J, Dupont, C, Herfarth, C, von Knebel Doeberitz, M & Schmitz-Agheguian, G 1998, 'Optimized non-radioactive protein truncation test for mutation analysis of the adenomatous polyposis coli (APC) gene', Clinical Chemistry and Laboratory Medicine, vol. 36, no. 8, pp. 567-570. https://doi.org/10.1515/CCLM.1998.097
Kirchgesser, Michael ; Albers, Andreas ; Vossen, Rolf ; Den Dunnen, Johan ; Van Ommen, Gert Jan ; Gebert, Johannes ; Dupont, Cecile ; Herfarth, Christian ; von Knebel Doeberitz, Magnus ; Schmitz-Agheguian, Gudrun. / Optimized non-radioactive protein truncation test for mutation analysis of the adenomatous polyposis coli (APC) gene. In: Clinical Chemistry and Laboratory Medicine. 1998 ; Vol. 36, No. 8. pp. 567-570.
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