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dc.contributor.authorWee, Eugene JH
dc.contributor.authorRauf, Sakandar
dc.contributor.authorShiddiky, Muhammad JA
dc.contributor.authorDobrovic, Alexander
dc.contributor.authorTrau, Matt
dc.date.accessioned2018-03-20T12:31:15Z
dc.date.available2018-03-20T12:31:15Z
dc.date.issued2015
dc.identifier.issn0009-9147
dc.identifier.doi10.1373/clinchem.2014.227546
dc.identifier.urihttp://hdl.handle.net/10072/172429
dc.description.abstractBACKGROUND: DNA methylation is a potential source of disease biomarkers. Typically, methylation levels are measured at individual cytosine/guanine (CpG) sites or over a short region of interest. However, regions of interest often show heterogeneous methylation comprising multiple patterns of methylation (epialleles) on individual DNA strands. Heterogeneous methylation is largely ignored because digital methods are required to deconvolute these usually complex patterns of epialleles. Currently, only single-molecule approaches, such as next generation sequencing (NGS), can provide detailed epiallele information. Because NGS is not yet feasible for routine practice, we developed a single-molecule–like approach, named for epiallele quantification (EpiQ). METHODS: EpiQ uses DNA ligases and the enhanced thermal instability of short (≤19 bases) mismatched DNA probes for the relative quantification of epialleles. The assay was developed using fluorescent detection on a gel and then adapted for electrochemical detection on a microfabricated device. NGS was used to validate the analytical accuracy of EpiQ. RESULTS: In this proof of principle study, EpiQ detected with 90%–95% specificity each of the 8 possible epialleles for a 3-CpG cluster at the promoter region of the CDKN2B (p15) tumor suppressor gene. EpiQ successfully profiled heterogeneous methylation patterns in clinically derived samples, and the results were cross-validated with NGS. CONCLUSIONS: EpiQ is a potential alternative tool for characterizing heterogeneous methylation, thus facilitating its use as a biomarker. EpiQ was developed on a gel-based assay but can also easily be adapted for miniaturized chip-based platforms.
dc.description.peerreviewedYes
dc.languageEnglish
dc.publisherAmerican Association for Clinical Chemistry (AACC)
dc.relation.ispartofpagefrom163
dc.relation.ispartofpageto171
dc.relation.ispartofissue1
dc.relation.ispartofjournalClinical Chemistry
dc.relation.ispartofvolume61
dc.subject.fieldofresearchMedical biotechnology
dc.subject.fieldofresearchMedical biotechnology not elsewhere classified
dc.subject.fieldofresearchMedical biochemistry and metabolomics
dc.subject.fieldofresearchClinical sciences
dc.subject.fieldofresearchcode3206
dc.subject.fieldofresearchcode320699
dc.subject.fieldofresearchcode3205
dc.subject.fieldofresearchcode3202
dc.titleDNA Ligase-Based Strategy for Quantifying Heterogeneous DNA Methylation without Sequencing
dc.typeJournal article
dc.type.descriptionC1 - Articles
dc.type.codeC - Journal Articles
gro.rights.copyrightSelf-archiving of the author-manuscript version is not yet supported by this journal. Please refer to the journal link for access to the definitive, published version or contact the author[s] for more information.
gro.hasfulltextNo Full Text
gro.griffith.authorShiddiky, Muhammad J.


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