The protein truncation test is based on the principle that stop codons generated by point mutations or frameshift mutations lead to a premature stop of translation. The coding region of the gene is amplified by PCR, using a sense primer tailed by a T7 promoter sequence. The amplified PCR product is then used as a template for in vitro translation testing. The PTT selectively detects translation-terminating mutations as short peptides. The size of the peptides is determined visually after autoradiography on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. It can be anticipated that small differences in the size of the protein fragments created by the truncating mutations may limit applications of the assay. For instance, truncated variants located close to the 5' binding site that result in very short translation products might be undetectable. Alternatively, for mutations situated close to the 3' binding site, the truncated and full-length products would be of nearly equal lengths and might not be resolved.
The PTT would be expected to be useful in the diagnosis of disorders to which terminating mutations contribute substantially. It has been used to scan genes related to disease, including familial adenomatous polyposis, hereditary breast and ovarian cancer, Duchenne's muscular dystrophy, and ophthalmic disease.82 A modification of the standard PTT called digital protein truncation to increase its sensitivity to detect mutations was utilized to detect APC mutations in fecal DNA from patients with colorectal tumors. Mutations were identified in 26 of 46 stool samples tested from patients with neoplastic disease, whereas none was identified in stools from 28 control patients who did not have neoplastic disease.83
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