Introduction of a myc reporter tag to improve the quality of mutation detection using the protein truncation test.
The protein truncation test is a useful method for identifying frameshift and nonsense mutations. The interpretation of real results is often made difficult by the presence of internally translated protein products and other nonspecific bands. To overcome these problems, the technique has been modified to include a myc reporter tag in the 5' primer used to amplify the genomic region of interest. The myc tag is recognised by a monoclonal antibody, which can be used to manipulate the products of the protein truncation test using immunoprecipitation techniques. The presence of the myc tag also introduces a mechanism whereby the use of radioisotopes can be avoided, relying instead on the identification of newly synthesised proteins using Western blot technology. The myc-tag modification can be integrated into all protein truncation tests, regardless of the gene being examined, with only one monoclonal antibody required for the immunoprecipitation purification and Western blotting.
Rowan
A J
AJ
Cancer Genetics Laboratory, Imperial Cancer Research Fund, London, UK.
Bodmer
W F
WF
D000911:Antibodies, Monoclonal; D001483:Base Sequence; D015153:Blotting, Western; D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers; D017930:Genes, Reporter; D016259:Genes, myc / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009841:Oligonucleotides; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D014176:Protein Biosynthesis; D016271:Proto-Oncogene Proteins c-myc / Q000276:immunology*; D014158:Transcription, Genetic
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D009841:Oligonucleotides; D016271:Proto-Oncogene Proteins c-myc