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dc.contributor.authorRand, Keith N
dc.contributor.authorYoung, Graeme Paul
dc.contributor.authorHo, Thu
dc.contributor.authorMolloy, Peter L
dc.date.accessioned2013-08-07T01:24:46Z
dc.date.available2013-08-07T01:24:46Z
dc.date.issued2012-09-10
dc.identifier.citationRand, K.N., Young, G.P., Ho, T. and Molloy, P.L., 2012. Sensitive and selective amplification of methylated DNA sequences using helper-dependent chain reaction in combination with a methylation-dependent restriction enzyme. Nucleic Acids Research, 41(1), e15.en
dc.identifier.issn0305-1048
dc.identifier.urihttp://hdl.handle.net/2328/26910
dc.description.abstractWe 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.en
dc.description.sponsorshipFunding for open access charge: Commonwealth Scientific and Industrial Research Organisation (CSIRO), Australia. National Health & Medical Research Councilen
dc.language.isoen
dc.publisherOxford University Pressen
dc.relationhttp://purl.org/au-research/grants/NHMRC/1006242en
dc.rightsThis is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.en
dc.subjectPublic healthen
dc.subjectCancer researchen
dc.subjectColorectal canceren
dc.titleSensitive and selective amplification of methylated DNA sequences using helper-dependent chain reaction in combination with a methylation-dependent restriction enzymeen
dc.typeArticleen
dc.relation.grantnumberNHMRC/1006242en
dc.identifier.doihttps://doi.org/10.1093/nar/gks831en
dc.rights.holderAuthors retain copyright.en
dc.rights.licenseCC-BY-NC


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