Allele-specific PCR analysis of p53 codon 249 AGT transversion in liver tissues from patients with viral hepatitis

Int J Cancer. 1996 Sep 27;68(1):21-5. doi: 10.1002/(SICI)1097-0215(19960927)68:1<21::AID-IJC4>3.0.CO;2-Z.


AGG to AGT mutations in codon 249 of the p53 tumor-suppressor gene are frequently observed in hepatocellular carcinomas (HCC) from areas where exposure to aflatoxin B1 (AFB) occurs. We developed a sensitive allele-specific polymerase chain reaction (AS-PCR) assay to detect this point mutation in non-neoplastic human liver tissues. Three oligonucleotide primers, 1 specific for the mutant allele and 2 specific for the wild-type allele were used. The mutant allele primer differed from the wild-type allele due to a G-to-T transversion in its terminal 3' nucleotide. The first stage involved amplification of exon 7 of p53 followed by a selective amplification of mutant codon 249 sequences. This method allowed for the detection of a mutant codon 249 allele in the presence of as many as 105 copies of the wild-type allele and was 100-fold more sensitive than the restriction fragment length polymorphism-PCR technique. We have applied this AS-PCR protocol to examine codon 249 AGT transversion in tumor and matched non-tumor liver samples from North American patients with hepatitis and from Mozambiquan patients exposed to AFB. Mutations were detected in 5 of 6 samples of non-neoplastic liver from Mozambiquan patients, all of whom were HBsAg- or HBcAg-positive and AFB-exposed. In contrast, no mutations were detected in non-neoplastic liver from North American patients with either HBV- or HCV-derived hepatitis and cirrhosis. This procedure is a simple and powerful approach for screening p53 codon 249 AGT mutation in heterogeneous non-neoplastic hepatocyte populations.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Aflatoxin B1 / adverse effects
  • Aged
  • Alleles*
  • Base Sequence
  • Codon*
  • Female
  • Genes, p53*
  • Hepatitis B / genetics*
  • Humans
  • Liver / chemistry*
  • Liver Neoplasms / chemically induced
  • Liver Neoplasms / genetics
  • Male
  • Middle Aged
  • Molecular Sequence Data
  • Mozambique
  • Point Mutation
  • Polymerase Chain Reaction*
  • Polymorphism, Restriction Fragment Length


  • Codon
  • Aflatoxin B1