Enrichment polymerase chain reaction for the detection of Ki-ras mutations: relevance of Taq polymerase error rate, initial DNA copy number, and reaction conditions on the emergence of false-positive mutant bands

Screening for oncogene mutations as a marker for malignancy can be a powerf ul tool for the early diagnosis of cancer. The enrichment polymerase chain reaction (PCR) is a sensitive method for the detection of low-frequency mut ations in small samples. However, false-positive results, caused by methodo logical errors, may have severe clinical implications. When applied to the detection of Ki-ras mutations in pancreatic secretions, the: assay sensitiv ity is limited to approximately 1:1400. Our investigation of Ki-ras mutatio ns in blood samples from patients with pancreatic carcinoma revealed PCR ba nds presumably derived from mutant Ki-ras in samples from healthy volunteer s, while all blood samples of the patients with pancreatic carcinomas showe d a wild-type band pattern. Mathematical modeling of the PCR reaction revea ls that the rate of false positive PCR results depends on the initial amoun t of DNA, the Tag polymerase error rater the number of PCR reaction cycles, reaction efficiency and the restriction endonuclease chosen. The overall e rror rate of false positive results of the enrichment PCR can be reduced to the square of the rate of a single-step analysis if repeated amplification s of the same DNA specimen show an identical result.