A locked nucleic acid clamp-mediated PCR assay for detection of a p53 codon 249 hotspot mutation in urine

Selena Y. Lin, Veerpal Dhillon, Surbhi Jain, Ting Tsung Chang, Chi Tan Hu, Yih Jyh Lin, Shun Hua Chen, Kung Chao Chang, Wei Song, Lixin Yu, Timothy M. Block, Ying Hsiu Su

Research output: Contribution to journalArticlepeer-review

19 Citations (Scopus)

Abstract

Hepatocellular carcinoma (HCC) has a 5-year survival rate of <10% because it is difficult to diagnose early. Mutations in the TP53 gene are associated with approximately 50% of human cancers. A hotspot mutation, a G:C to T:A transversion at codon 249 (249T), may be a potential DNA marker for HCC screening because of its exclusive presence in HCC and its detection in the circulation of some patients with HCC. A locked nucleic acid clamp-mediated PCR assay, followed by melting curve analysis (using the SimpleProbe), was developed to detect the TP53 249T mutation. In this assay, the locked nucleic acid clamp suppressed 107 copies of wild-type templates and permitted detection of 249Tmutated template, with a sensitivity of 0.1% (1:1000) of the mutant/wild-type ratio, assessed by a reconstituted standard within 2 hours. With an amplicon size of 41 bp, it detects target DNA sequences in short fragmented DNA templates. The detected mutations were validated by DNA sequencing analysis. We then tested DNA isolated from urine samples of patients with HCC for p53 mutations and identified positive TP53 mutations in 9 of 17 samples. The possibility of using this novel TP53 249T assay to develop a urine or blood test for HCC screening is discussed.

Original languageEnglish
Pages (from-to)474-484
Number of pages11
JournalJournal of Molecular Diagnostics
Volume13
Issue number5
DOIs
Publication statusPublished - 2011 Sep

All Science Journal Classification (ASJC) codes

  • Pathology and Forensic Medicine
  • Molecular Medicine

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