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Related Experiment Video

Updated: Jun 8, 2026

Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR
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COLD PCR HRM: a highly sensitive detection method for IDH1 mutations.

Blandine Boisselier1, Yannick Marie, Marianne Labussière

  • 1Université Pierre et Marie Curie-Paris 6, Centre de Recherche de l'Institut du Cerveau et de la Moëlle épinière (CRICM) UMR-S975, Paris, France.

Human Mutation
|October 2, 2010
PubMed
Summary

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Coamplification at lower temperature-PCR (COLD PCR) combined with high-resolution melting (HRM) significantly enhances detection of the IDH1(R132H) mutation in gliomas. This method is 100-fold more sensitive than Sanger sequencing, especially in contaminated samples.

Area of Science:

  • Oncology
  • Molecular Biology
  • Genetics

Background:

  • The isocitrate dehydrogenase 1 (IDH1) p.Arg132His mutation is a key prognostic marker in gliomas.
  • Direct sequencing struggles to detect this mutation in samples with high levels of normal DNA contamination.

Purpose of the Study:

  • To evaluate the sensitivity of coamplification at lower temperature-PCR (COLD PCR) combined with high-resolution melting (HRM) for IDH1(R132H) mutation detection.
  • To assess the utility of COLD PCR-HRM for analyzing highly contaminated tumor samples.

Main Methods:

  • Serial dilutions of mutant and wild-type DNA were used to test PCR-HRM sensitivity.
  • Coamplification at lower temperature-PCR (COLD PCR) was applied in one or two runs.
  • High-resolution melting (HRM) analysis was performed after COLD PCR.

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  • Tumor edge biopsies, histologically tumor-free, were analyzed using immunohistochemistry and double COLD PCR-HRM.

    • Main Results:

    • Standard PCR-HRM detected IDH1(R132H) at 25% abundance, similar to Sanger sequencing.
    • A single COLD PCR run improved detection to 2% mutant DNA.
    • Two consecutive COLD PCR runs detected 0.25% mutant DNA, mimicking highly contaminated samples.
    • Immunohistochemistry identified the mutation in 30% of tumor edge biopsies, while double COLD PCR-HRM detected it in 100%.

    Conclusions:

    • COLD PCR-HRM offers a 100-fold increase in sensitivity compared to Sanger sequencing for IDH1(R132H) detection.
    • This method is highly effective for analyzing tumor samples with significant normal DNA contamination.
    • COLD PCR-HRM is a rapid and powerful strategy for sensitive glioma mutation analysis.