Journal article
A locked nucleic acid clamp-mediated PCR assay for detection of a p53 codon 249 hotspot mutation in urine
The Journal of molecular diagnostics : JMD, Vol.13(5), pp.474-484
Sep 2011
PMCID: PMC3157622
PMID: 21726666
Featured in Collection : UN Sustainable Development Goals @ Drexel
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 10(7) copies of wild-type templates and permitted detection of 249T-mutated 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.
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Details
- Title
- A locked nucleic acid clamp-mediated PCR assay for detection of a p53 codon 249 hotspot mutation in urine
- Creators
- Selena Y Lin - Drexel UniversityVeerpal Dhillon - Drexel UniversitySurbhi Jain - Drexel UniversityTing-Tsung Chang - National Cheng Kung UniversityChi-Tan Hu - Tzu Chi UniversityYih-Jyh Lin - National Cheng Kung University HospitalShun-Hua Chen - National Cheng Kung UniversityKung-Chao Chang - National Cheng Kung UniversityWei Song - JBS ScienceLixin Yu - Drexel UniversityTimothy M Block - Drexel UniversityYing-Hsiu Su - Drexel University
- Publication Details
- The Journal of molecular diagnostics : JMD, Vol.13(5), pp.474-484
- Publisher
- Elsevier
- Grant note
- R01 CA125642 / NCI NIH HHS CA93176 / NCI NIH HHS
- Resource Type
- Journal article
- Language
- English
- Academic Unit
- A.J. Drexel Autism Institute; Microbiology and Immunology
- Identifiers
- 991019167539804721
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- Domestic collaboration
- International collaboration
- Web of Science research areas
- Pathology