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 potentially useful as sensitive tests for tumor diagnosis. The technical challenge 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 determined 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 273). Depending on the identity of the mismatch, mismatched template was amplified 10(2)-10(4)-fold less than perfectly matched template. In other words, a mutant allele could be detected by ASA if it represented > 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 amplification of mutant sequences was achieved using a PNA complementary to the normal sequence to inhibit the amplification of wild-type DNA. PNA clamping followed by ASA resulted in significant improvement in sensitivity and specificity, permitting the detection of tumor DNA diluted with a 300,000-fold excess of normal human DNA.
Rhodes
C H
CH
Department of Pathology, Dartmouth Hitchcock Medical Center, Lebanon, New Hampshire 03756, USA.
Honsinger
C
C
Porter
D M
DM
Sorenson
G D
GD
D000483:Alleles; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics; D016158:Genes, p53; D011905:Genes, ras; D006801:Humans; D009154:Mutation; D009369:Neoplasms / Q000175:diagnosis* / Q000235:genetics*; D010446:Peptide Fragments / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D019914:Taq Polymerase; D013698:Templates, Genetic
D017931:DNA Primers; D010446:Peptide Fragments; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase