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).
CITATION STYLE
Candiloro, I. L. M., Mikeska, T., & Dobrovic, A. (2011). Closed-Tube PCR Methods for Locus-Specific DNA Methylation Analysis. In Methods in Molecular Biology (Vol. 791, pp. 55–71). Humana Press Inc. https://doi.org/10.1007/978-1-61779-316-5_5
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