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PCR-Based Methods for Mutation Detection

  • Chapter
Molecular Diagnostics

Abstract

Although many, even most, methods of mutation detection depend on polymerase chain reaction (PCR), in the majority of techniques PCR itself does not detect the actual mutation. Rather, PCR generates an amplicon that is then analyzed by some other method to find possible mutations within the ampli-con, such as conformation-based techniques like single-stranded conformational polymorphism (SSCP) analysis, denaturing gradient gel electrophoresis (DGGE), or sequencing. However, there are some methods in which a modified PCR acts as the primary mutation-detection system, although some type of electrophoresis might be needed to separate the subsequent amplicons. These include real-time PCR, the amplification refractory mutation system (ARMS), quantitative fluorescent PCR (QF-PCR), or a derivative of the oligoligation assay, multiplex ligation-dependent probe amplification (MLPA). Also discussed is the single-nucleotide primer extension assay and a proprietary derivative of it called Pronto™. Because it uses a DNA polymerase in a post-PCR extension step, it can be deemed to fall into the group of PCR-based methods of mutation detection. A primary limitation of these methods is that, with a few exceptions, they are only suitable for testing for mutations that have been previously detected and characterized by other techniques.

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Author information

Authors and Affiliations

  1. Clinical Genetics Laboratory, Institute of Medical Genetics, University Hospital of Wales, Cardiff, UK

    Ian M. Frayling MA, MB, BChir, PhD, MRCPath

  2. East Anglian Regional Genetics Service, Molecular Genetics Laboratory, Addenbrooke’s Hospital, Cambridge, UK

    Emma Monk BSc

  3. Molecular Genetics Laboratory, Institute of Medical Genetics, Cardiff, UK

    Rachel Butler BSc(Hons), MRCPath

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  1. Ian M. Frayling MA, MB, BChir, PhD, MRCPath
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  2. Emma Monk BSc
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  3. Rachel Butler BSc(Hons), MRCPath
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Editors and Affiliations

  1. Department of Pathology and Laboratory Medicine, University of North Carolina School of Medicine, Chapel Hill, NC, USA

    William B. Coleman

  2. Department of Pathology, Dartmouth-Hitchcock Medical Center, Lebanon, NH, USA

    Gregory J. Tsongalis

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© 2006 Humana Press, a part of Springer Science+Business Media, LLC

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Frayling, I.M., Monk, E., Butler, R. (2006). PCR-Based Methods for Mutation Detection. In: Coleman, W.B., Tsongalis, G.J. (eds) Molecular Diagnostics. Humana Press. https://doi.org/10.1385/1-59259-928-1:065

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  • DOI: https://doi.org/10.1385/1-59259-928-1:065

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