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COLD-PCR Technologies in the Area of Personalized Medicine: Methodology and Applications

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Abstract

Somatic mutations bear great promise for use as biomarkers for personalized medicine, but are often present only in low abundance in biological material and are therefore difficult to detect. Many assays for mutation analysis in cancer-related genes (hotspots) have been developed to improve diagnosis, prognosis, prediction of drug resistance, and monitoring of the response to treatment. Two major approaches have been developed: mutation-specific amplification methods and methods that enrich and detect mutations without prior knowledge on the exact location and identity of the mutation. CO-amplification at Lower Denaturation temperature Polymerase Chain Reaction (COLD-PCR) methods such as full-, fast-, ice- (improved and complete enrichment), enhanced-ice, and temperature-tolerant COLD-PCR make use of a critical temperature in the polymerase chain reaction to selectively denature wild-type-mutant heteroduplexes, allowing the enrichment of rare mutations. Mutations can subsequently be identified using a variety of laboratory technologies such as high-resolution melting, digital polymerase chain reaction, pyrosequencing, Sanger sequencing, or next-generation sequencing. COLD-PCR methods are sensitive, specific, and accurate if appropriately optimized and have a short time to results. A large variety of clinical samples (tumor DNA, circulating cell-free DNA, circulating cell-free fetal DNA, and circulating tumor cells) have been studied using COLD-PCR in many different applications including the detection of genetic changes in cancer and infectious diseases, non-invasive prenatal diagnosis, detection of microorganisms, or DNA methylation analysis. In this review, we describe in detail the different COLD-PCR approaches, highlighting their specificities, advantages, and inconveniences and demonstrating their use in different fields of biological and biomedical research.

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Acknowledgements

The authors thank Steven McGinn (Centre National de Génotypage) for critical reading of the manuscript.

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FM, AH, and JT reviewed the literature and wrote the manuscript.

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    1. Laboratory for Epigenetics and Environment, Centre National de Génotypage, CEA-Institut de Génomique, Batiment G2, 2 rue Gaston Crémieux, 91000, Evry, France

      Florence Mauger & Jörg Tost

    2. Laboratory for Genomics, Fondation Jean Dausset-CEPH, 75010, Paris, France

      Alexandre How-Kit

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    Correspondence to Jörg Tost.

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    Work in the laboratory of JT is supported by Grants from the Agence Nationale de Recherche (ANR-13-EPIG-0003-05 and ANR-13-CESA-0011-05), Aviesan/INSERM (EPIG2014-01 and EPlG2014-18), and INCa (PRT-K14-049), a Sirius research award (UCB Pharma S.A.), a Passerelle research award (Pfizer), iCARE (MSD Avenir), and the institutional budget of the Centre National de Génotypage.

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    Florence Mauger, Alexandre How-Kit, and Jörg Tost declare no conflicts of interest.

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    F. Mauger and A. How-Kit contributed equally and should be considered as joint first authors.

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    Mauger, F., How-Kit, A. & Tost, J. COLD-PCR Technologies in the Area of Personalized Medicine: Methodology and Applications. Mol Diagn Ther 21, 269–283 (2017). https://doi.org/10.1007/s40291-016-0254-8

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