Allele-specific PCR analysis of p53 codon 249 AGT transversion in liver tissues from patients with viral hepatitis
✍ Scribed by Gordon M. Kirby; Gerald Batist; Nasser Fotouhi-Ardakani; Hisayoshi Nakazawa; Hiroshi Yamasaki; Michael Kew; Ross G. Cameron; Moulay A. Alaoui-Jamali
- Publisher
- John Wiley and Sons
- Year
- 1996
- Tongue
- French
- Weight
- 684 KB
- Volume
- 68
- Category
- Article
- ISSN
- 0020-7136
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✦ Synopsis
AGG to AGT mutations in codon 249 of the p53 tumorsuppressor ene are frequently observed in hepatocellular carcinomas ~HCC) from areas where exposure to anatoxin B, (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, I 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 invoked amplification of =on 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 10s copies of the wild-allele and was 106-fold more sensitive than the restriction%gment length polymorphism-PCR techni ue. We have applied this AS-PCR protocol to examine codon 248 AGT transversion in tumor and matched non-tumor liver samples from North American patients with hepatitis and from Mozambi uan patients exposed to AFB. Mutations were detected i n 1 of 6 samples of non-neoplastic liver from Mozambican patients, all of whom were HBsAgor HBcAg-positive and AFB-exposed. In contrast, no mutations were detected in non-neaplastic liver from North American patients with either HBV-or HCV-derived hepatitis and cirrhosis. This rocedure is a simple and powerful approach for screening pS! codon 249 AGT mutation in heterogeneous non-neoplastic hepatoqte 3~ Wiley-Liss, Inc.
ulatiom.
MATERIAL AND METHODS
Source and isolation of human DNA
Liver samples collected from hepatitis patients from Mozambique, Canada and the United States were snap-frozen in liquid nitrogen immediately following surgical removal. Histopathological analysis of liver tumors and surrounding nonneoplastic liver were assessed independently by several pathologists. Genomic DNA was isolated from neoplastic and nonneoplastic liver by phenol/chloroform extraction and quantified 6To whom correspondence and reprint requests should be sent, at