title: A locked nucleic acid clamp-mediated PCR assay for detection of a p53 codon 249 hotspot mutation in urine.
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.
authors: Department of Microbiology and Immunology, Drexel University College of Medicine, Doylestown, Pennsylvania, USA.
authors: Selena Y
authors: SY
authors: Lin
authors: Veerpal
authors: V
authors: Dhillon
authors: Surbhi
authors: S
authors: Jain
authors: Ting-Tsung
authors: TT
authors: Chang
authors: Chi-Tan
authors: CT
authors: Hu
authors: Yih-Jyh
authors: YJ
authors: Lin
authors: Shun-Hua
authors: SH
authors: Chen
authors: Kung-Chao
authors: KC
authors: Chang
authors: Wei
authors: W
authors: Song
authors: Lixin
authors: L
authors: Yu
authors: Timothy M
authors: TM
authors: Block
authors: Ying-Hsiu
authors: YH
authors: Su
mesh_terms: D000328:Adult; D000368:Aged; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000652:urine; D003062:Codon / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D015342:DNA Probes / Q000378:metabolism; D005260:Female; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics / Q000652:urine; D008297:Male; D008875:Middle Aged; D009154:Mutation / Q000235:genetics*; D009691:Nucleic Acid Denaturation / Q000235:genetics; D009841:Oligonucleotides / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D016159:Tumor Suppressor Protein p53 / Q000235:genetics* / Q000652:urine*
chemical_list: D003062:Codon; D015342:DNA Probes; D009841:Oligonucleotides; D016159:Tumor Suppressor Protein p53; C477371:locked nucleic acid