title: Closed-tube PCR methods for locus-specific DNA methylation analysis.
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).
authors.affiliation: Molecular Pathology Research and Development Laboratory, Department of Pathology, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia.
authors.forename: Ida L M
authors.initials: IL
authors.lastname: Candiloro
authors.forename: Thomas
authors.initials: T
authors.lastname: Mikeska
authors.forename: Alexander
authors.initials: A
authors.lastname: Dobrovic
mesh_terms: D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D019175:DNA Methylation / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D056426:Genetic Loci / Q000235:genetics*; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods* / Q000592:standards; D012015:Reference Standards; D013447:Sulfites / Q000494:pharmacology; D013997:Time Factors
chemical_list: D017931:DNA Primers; D013447:Sulfites; D004247:DNA; C042345:hydrogen sulfite