Analysis of the allele-specific PCR method for the detection of neoplastic disease

Diagn Mol Pathol. 1997 Feb;6(1):49-57. doi: 10.1097/00019606-199702000-00008.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alleles*
  • DNA Primers
  • DNA-Directed DNA Polymerase / chemistry
  • DNA-Directed DNA Polymerase / genetics
  • Genes, p53
  • Genes, ras
  • Humans
  • Mutation
  • Neoplasms / diagnosis*
  • Neoplasms / genetics*
  • Peptide Fragments / genetics
  • Polymerase Chain Reaction / methods*
  • Taq Polymerase
  • Templates, Genetic


  • DNA Primers
  • Peptide Fragments
  • Taq Polymerase
  • DNA-Directed DNA Polymerase