title: Sensitive and selective amplification of methylated DNA sequences using helper-dependent chain reaction in combination with a methylation-dependent restriction enzymes.
abstract: We have developed a novel technique for specific amplification of rare methylated DNA fragments in a high background of unmethylated sequences that avoids the need of bisulphite conversion. The methylation-dependent restriction enzyme GlaI is used to selectively cut methylated DNA. Then targeted fragments are tagged using specially designed 'helper' oligonucleotides that are also used to maintain selection in subsequent amplification cycles in a process called 'helper-dependent chain reaction'. The process uses disabled primers called 'drivers' that can only prime on each cycle if the helpers recognize specific sequences within the target amplicon. In this way, selection for the sequence of interest is maintained throughout the amplification, preventing amplification of unwanted sequences. Here we show how the method can be applied to methylated Septin 9, a promising biomarker for early diagnosis of colorectal cancer. The GlaI digestion and subsequent amplification can all be done in a single tube. A detection sensitivity of 0.1% methylated DNA in a background of unmethylated DNA was achieved, which was similar to the well-established Heavy Methyl method that requires bisulphite-treated DNA.
authors.affiliation: CSIRO Animal, Food and Health Sciences, Preventative Health Flagship, North Ryde, NSW 1670, Australia. keith.rand@csiro.au
authors.forename: Keith N
authors.initials: KN
authors.lastname: Rand
authors.forename: Graeme P
authors.initials: GP
authors.lastname: Young
authors.forename: Thu
authors.initials: T
authors.lastname: Ho
authors.forename: Peter L
authors.initials: PL
authors.lastname: Molloy
mesh_terms: D001483:Base Sequence; D015179:Colorectal Neoplasms / Q000235:genetics; D019175:DNA Methylation; D004262:DNA Restriction Enzymes; D006801:Humans; D020014:K562 Cells; D009841:Oligonucleotides; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D058112:Septins / Q000235:genetics
chemical_list: D009841:Oligonucleotides; D004262:DNA Restriction Enzymes; C474315:SEPTIN9 protein, human; D058112:Septins