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LT-RPA: An Isothermal DNA Amplification Approach for Improved Microsatellite Genotyping and Microsatellite Instability Detection

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Clinical Applications of Nucleic Acid Amplification

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2621))

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Abstract

Microsatellites are short tandem repeats of one to six nucleotides that are highly polymorphic and extensively used as genetic markers in numerous biomedical applications, including the detection of microsatellite instability (MSI) in cancer. The standard analytical method for microsatellite analysis relies on PCR amplification followed by capillary electrophoresis or, more recently, next-generation sequencing (NGS). However, their amplification during PCR generates undesirable frameshift products known as stutter peaks caused by polymerase slippage, complicating data analysis and interpretation, while very few alternative methods for microsatellite amplification have been developed to reduce the formation of these artifacts. In this context, the recently developed low-temperature recombinase polymerase amplification (LT-RPA) is an isothermal DNA amplification method at low temperature (32 °C) that drastically reduces and sometimes completely abolishes the formation of stutter peaks. LT-RPA greatly simplifies the genotyping of microsatellites and improves the detection of MSI in cancer. In this chapter, we describe in detail all the experimental steps necessary for the development of LT-RPA simplex and multiplex assays for microsatellite genotyping and MSI detection, including the design, optimization, and validation of the assays combined with capillary electrophoresis or NGS.

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  • 23 June 2023

    A correction has been published.

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Acknowledgments

We want to thank Steven McGinn (CNRGH) for his careful editing of the manuscript and improvement of the English. This work has been supported by the Fondation ARC pour la recherche sur le cancer (PJA 20191209442). LMH received support from the GENMED Laboratory of Excellence on Medical Genomics [ANR-10-LABX-0013]. SIJ and VR contributed equally to this work.

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Author notes

  1. The authors “Sophie I. Jeanjean” and “Victor Renault” contributed equally to this chapter.

Authors and Affiliations

  1. Laboratoire de Génomique, Fondation Jean Dausset – CEPH, Paris, France

    Sophie I. Jeanjean, Antoine Daunay, Yimin Shen, Lise M. Hardy, Jean-François Deleuze & Alexandre How-Kit

  2. Laboratoire de Bio-informatique Clinique, Institut Curie, Paris, France

    Victor Renault

  3. Laboratory of Excellence GenMed, Paris, France

    Lise M. Hardy & Jean-François Deleuze

  4. Centre National de Recherche en Génomique Humaine, CEA- Institut François Jacob, Evry, France

    Jean-François Deleuze

Authors
  1. Sophie I. Jeanjean
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  2. Victor Renault
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  3. Antoine Daunay
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  4. Yimin Shen
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  5. Lise M. Hardy
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  6. Jean-François Deleuze
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  7. Alexandre How-Kit
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Corresponding author

Correspondence to Alexandre How-Kit .

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Editors and Affiliations

  1. Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR, USA

    Meagan B. Myers

  2. Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, SC, USA

    Cynthia A. Schandl

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Jeanjean, S.I. et al. (2023). LT-RPA: An Isothermal DNA Amplification Approach for Improved Microsatellite Genotyping and Microsatellite Instability Detection. In: Myers, M.B., Schandl, C.A. (eds) Clinical Applications of Nucleic Acid Amplification. Methods in Molecular Biology, vol 2621. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2950-5_7

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  • DOI: https://doi.org/10.1007/978-1-0716-2950-5_7

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2949-9

  • Online ISBN: 978-1-0716-2950-5

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