A non-radioactive protein truncation test for the sensitive detection of all stop and frameshift mutations.
A new method for mutation detection is described, which is a technical advancement of the protein truncation test. The new technique is non-radioactive and highly sensitive for detection of virtually all sequence mutations, which lead to a stop signal or to the shift of the translation frame. The method includes four steps: 1) capture of the interesting sequence copies out of the sample by binding to an immobilized complementary sequence, 2) PCR amplification of the gene fragment to be analyzed with primers coding both for amino- and carboxy-terminal tags, 3) in vitro transcription and translation, and 4) analysis of the translation products by Western blot. As an evaluation of the new method, we detected mutated gene copies at a dilution of 1 to 40 compared to the non-mutated gene. Using the method, we were able to detect a mutation in the adenomatous polyposis coli tumor suppressor gene (APC) in a stool sample of a colorectal cancer patient. This mutation could not be detected by direct sequencing of the amplified APC gene fragment.
Kahmann
Sabine
S
Max-Planck-Institut für molekulare Physiologie, Dortmund, Germany.
Herter
Peter
P
Kuhnen
Cornelius
C
Müller
Klaus-Michael
KM
Muhr
Gert
G
Martin
Dirk
D
Soddemann
Matthias
M
Müller
Oliver
O
D011125:Adenomatous Polyposis Coli / Q000235:genetics*; D025601:Adenomatous Polyposis Coli Protein / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D015153:Blotting, Western; D003062:Codon / Q000235:genetics; D018389:Codon, Nonsense / Q000235:genetics*; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004252:DNA Mutational Analysis / Q000379:methods; D005243:Feces / Q000737:chemistry; D016368:Frameshift Mutation / Q000235:genetics*; D017491:Genes, APC; D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011868:Radioisotopes; D012680:Sensitivity and Specificity; D013698:Templates, Genetic; D014158:Transcription, Genetic
D025601:Adenomatous Polyposis Coli Protein; D003062:Codon; D018389:Codon, Nonsense; D011868:Radioisotopes; D004247:DNA