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Title: DNA ligase-based strategy for quantifying heterogeneous DNA methylation without sequencing.
Austin Authors: Wee, Eugene J H;Rauf, Sakandar;Shiddiky, Muhammad J A;Dobrovic, Alexander ;Trau, Matt
Department of Pathology, University of Melbourne, Parkville, Victoria, Australia
Translational Genomics & Epigenomics Laboratory, Ludwig Institute for Cancer Research, Olivia Newton-John Cancer & Wellness Centre, Heidelberg, Victoria, Australia
Centre for Personalized Nanomedicine, Australian Institute for Bioengineering and Nanotechnology (AIBN), and School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, Queensland, Australia
Centre for Personalized Nanomedicine, Australian Institute for Bioengineering and Nanotechnology (AIBN), and.
Issue Date: 1-Oct-2014
Publication information: Clinical Chemistry 2014; 61(1): 163-71
Abstract: 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).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.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.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.
Gov't Doc #: 25274555
DOI: 10.1373/clinchem.2014.227546
Type: Journal Article
Subjects: Alleles
CpG Islands.genetics
DNA Ligases.chemistry
DNA Methylation.genetics
Electrochemical Techniques
Electrophoresis, Polyacrylamide Gel
Epigenesis, Genetic
Genetic Heterogeneity
Ligase Chain Reaction
Molecular Diagnostic Techniques.instrumentation.methods
Polymerase Chain Reaction
Reproducibility of Results
Sensitivity and Specificity
Sequence Analysis, DNA
Appears in Collections:Journal articles

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