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Closed-Tube PCR Methods for Locus-Specific DNA Methylation Analysis

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Epigenetics Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 791))

Abstract

Closed-tube PCR methods (sometimes referred to as in-tube PCR methods) for locus-specific DNA ­methylation analysis are methodologies in which the amplification and analysis of bisulphite-modified DNA take place in one tube without the need to remove the PCR products for further analysis. Closed-tube methodologies lend themselves to high-throughput applications and molecular diagnostics but are also applicable as a research tool. We review three closed-tube methodologies, methylation-sensitive high-resolution melting (MS-HRM), MethyLight, and sensitive melting after real-time analysis – methylation-specific PCR (SMART-MSP). Closed-tube detection can be performed by simultaneously amplifying both methylated and unmethylated templates and subsequent melting curve analysis (MS-HRM). Alternatively, methylation-specific primers are used in real-time quantitative PCR and monitored either by a fluorescent hydrolysis probe (MethyLight) or using a double-stranded DNA binding fluorescent dye with a subsequent quality control step by melting curve analysis (SMART-MSP).

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Acknowledgements

AD has received grant support from the Cancer Council of Victoria, the National Breast Cancer Foundation of Australia, the Victorian Cancer Agency, the National Health and Medical Research Council of Australia, the Susan G. Komen for the Cure Foundation, and the US Department of Defence Breast Cancer Research Program under award numbers W81XWH-05-1-0500 and W81XWH-06-1-0670. Views and opinions of, and endorsements by the authors do not reflect those of the US Army or the Department of Defence.

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Authors and Affiliations

  1. Molecular Pathology Research and Development Laboratory, Department of Pathology, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia

    Ida L. M. Candiloro, Thomas Mikeska & Alexander Dobrovic

Authors
  1. Ida L. M. Candiloro
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  2. Thomas Mikeska
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  3. Alexander Dobrovic
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Correspondence to Alexander Dobrovic .

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Editors and Affiliations

  1. Dept. Biology, University of Alabama, Birmingham, University Blvd. 1300, Birmingham, 35294-1170, Alabama, USA

    Trygve O. Tollefsbol

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Candiloro, I.L.M., Mikeska, T., Dobrovic, A. (2011). Closed-Tube PCR Methods for Locus-Specific DNA Methylation Analysis. In: Tollefsbol, T. (eds) Epigenetics Protocols. Methods in Molecular Biology, vol 791. Humana Press. https://doi.org/10.1007/978-1-61779-316-5_5

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  • DOI: https://doi.org/10.1007/978-1-61779-316-5_5

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-315-8

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