Publications & Reports

Closed-tube PCR methods for locus-specific DNA methylation analysis.

Ida L M Candiloro, Thomas Mikeska, Alexander Dobrovic
Molecular Pathology Research and Development Laboratory, Department of Pathology, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia.


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).


  • Journal: Methods in Molecular Biology
  • Published: 01/07/2011
  • Volume: 791
  • Pagination: 55-71