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.

Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1

Detection of p53 codon 249 mutations by an LNA clamp-mediated PCR assay and melting curve analysis with SimpleProbes. A: Locations and sequences of primers (arrows), clamp (underlined), and probes (shaded) used in the assay. Codons 248 and 249 are indicated by vertical lines. LNAs are italicized. SimpleProbe sequences shown with LNA are italicized and capitalized. PCR products derived from plasmids p53 WT, p53_249T, p53_249C, or H2O (arrows) were determined by melting curve analysis with the SimpleProbes 249WT (B), 249T (C), and 249C (D).

Figure 2

Selective suppression of PCR amplification of TP53 WT templates by the LNA clamp. Melting curve analysis of the PCR products generated from the p53_WT (gray) or p53_249T (black) template in the absence (-) or presence (+) of the LNA clamp using SimpleProbe SP_249T. A and B: The PCR products were generated from a range of 101 to 107 copies of WT (gray) templates, with 10 copies of the p53_249T (black) template in each reaction. The ratios of p53_249T/p53_WT are indicated. C and D: Melting curve analysis of the PCR products generated from a range of 101 to 107 copies of the mutated p53_249T template with 107 copies of the p53_WT template. The ratios of p53_249T/p53_WT are indicated.

Figure 3

Comparison of melting curve analyses and DNA sequencing chromatograms of the HCC tissue DNA samples that contained the p53 mutation detectable by the p53_249 assay. Melting curves of the PCR products generated in the absence (-) or presence (+) of the LNA clamp using the SimpleProbe SP_249T are shown in A and B. The chromatograms of the sequence of interest, obtained by DNA sequencing analysis of the respective PCR clones, as described in the text, are shown in C. The sequences of the 249 codon are underlined, and the mutated or inserted sequences are boxed. In each reaction, p53_WT and p53_249T templates were used as controls.

Figure 4

Detection of p53 mutations in urine samples from patients with HCC. HMW and LMW urine DNA samples (U1 to U17), isolated as described in Materials and Methods, were subjected to the LNA clamp-mediated PCR assay for the p53 mutation. PCRs were performed in the absence (-) or presence (+) of the LNA clamp; the melting curve analysis using the SimpleProbe SP_249T followed. In each reaction, p53_WT and p53_249T templates were used as controls.

Figure 4

Detection of p53 mutations in urine samples from patients with HCC. HMW and LMW urine DNA samples (U1 to U17), isolated as described in Materials and Methods, were subjected to the LNA clamp-mediated PCR assay for the p53 mutation. PCRs were performed in the absence (-) or presence (+) of the LNA clamp; the melting curve analysis using the SimpleProbe SP_249T followed. In each reaction, p53_WT and p53_249T templates were used as controls.