title: Detection and analysis of DNA methylation by pyrosequencing.
abstract: Pyrosequencing is a "sequencing by synthesis" technique which can be used to quantify DNA methylation at specific CpG sites within the target region of interest. Biotin labelled polymerase chain reaction (PCR) products form the template for base-pair nucleotide incorporation causing a light emitting cascade reaction resulting in the formation of a pyrogram and the calculation of the percentage methylation for each site. Prior to pyrosequencing, it is essential to bisulphite-convert the DNA sample and then perform locus-specific PCR for the region of interest. One of the PCR primers needs to be biotinylated and a separate sequencing primer is required for the pyrosequencing itself.
authors.affiliation: Haematology, Centre for Cancer Research and Cell Biology Belfast, Queen's University Belfast, Belfast, Northern Ireland, UK. hcolyer01@qub.ac.uk
authors.forename: Hilary A A
authors.initials: HA
authors.lastname: Colyer
authors.forename: Richard N
authors.initials: RN
authors.lastname: Armstrong
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Sharpe
authors.forename: Ken I
authors.initials: KI
authors.lastname: Mills
mesh_terms: D001710:Biotin; D018899:CpG Islands / Q000235:genetics*; D003596:Cytosine / Q000737:chemistry; D019175:DNA Methylation / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D057890:Epigenomics / Q000379:methods*; D006801:Humans; D008156:Luciferases; D015394:Molecular Structure; D016133:Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D017931:DNA Primers; D001710:Biotin; D003596:Cytosine; D008156:Luciferases