ANALYSIS OF THE ALLELE-SPECIFIC PCR METHOD FOR THE DETECTION OF NEOPLASTIC DISEASE

PCR assays for the presence of mutant K-ras or p53 sequences are poten tially useful as sensitive tests for tumor diagnosis. The technical ch allenge is to design assays sensitive enough to detect a few molecules of mutant DNA yet sufficiently specific that a false positive signal is not produced by a 10(5)- or 10(6)-fold excess of normal DNA. We det ermined the detection limit of allele-specific PCR (ASA) as a function of the particular mismatch involved using all 12 possible mismatches in two different DNA sequence contexts (K-ras codon 12 and p53 codon 2 73). Depending on the identity of the mismatch, mismatched template wa s amplified 10(2)-10(4)-fold less than perfectly matched template. In other words, a mutant allele could be detected by ASA if it represente d >1-0.01% of the total DNA from that locus. Peptide nucleic acid (PNA ) clamping was used to improve the K-ras ASA assay. Selective amplific ation of mutant sequences was achieved using a PNA complementary to th e normal sequence to inhibit the amplification of wild-type DNA. PNA c lamping followed by ASA resulted in significant improvement in sensiti vity and specificity, permitting the detection of tumor DNA diluted wi th a 300,000-fold excess of normal human DNA.