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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,

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Presentation on theme: "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,"— Presentation transcript:

1 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  The Journal of Molecular Diagnostics  Volume 13, Issue 5, Pages (September 2011) DOI: /j.jmoldx Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

2 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). The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx ) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

3 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. The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx ) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

4 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. The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx ) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

5 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. The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx ) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

6 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. The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx ) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

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