title: Microdevices for detecting locus-specific DNA methylation at CpG resolution.
abstract: Simple, rapid, and inexpensive strategies for detecting DNA methylation could facilitate routine patient diagnostics. Herein, we describe a microdevice based electrochemical assay for the detection of locus-specific DNA methylation at single CpG dinucleotide resolution after bisulfite conversion of a target DNA sequence. This is achieved by using the ligase chain reaction (LCR) to recognize and amplify a C to T base change at a CpG site and measuring the change electrochemically (eLCR). Unlike other electrochemical detection methods for DNA methylation, methylation specific (MS)-eLCR can potentially interrogate any CpG of interest in the genome. MS-eLCR also distinguishes itself from other electrochemical LCR detection schemes by integrating a peroxidase-mimicking DNAzyme sequence into the LCR amplification probes design which in turn, serves as a redox moiety when bound with a hemin cofactor. Following hybridization to surface-bound capture probes, the DNAzyme-linked LCR products induce electrocatalytic responses that are proportional to the methylation levels of the gene locus in the presence of hydrogen peroxide. The performance of the assay was evaluated by simultaneously detecting C to T changes at a locus associated with cancer metastasis in breast cancer cell lines and serum-derived samples. MS-eLCR required as little as 0.04 pM of starting material and was sensitive to 10-15% methylation change with good reproducibility (RSD=7.9%, n=3). Most importantly, the accuracy of the method in quantifying locus-specific methylation was comparable to both fluorescence-based and Next Generation Sequencing approaches. We thus believe that the proposed assay could potentially be a low cost alternative to current technologies for DNA methylation detection of specific CpG sites.
authors: Centre for Biomarker Research and Development, Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, Brisbane, QLD 4072, Australia; School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD 4072, Australia.
authors: Kevin M
authors: KM
authors: Koo
authors: Centre for Biomarker Research and Development, Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, Brisbane, QLD 4072, Australia.
authors: Eugene J H
authors: EJ
authors: Wee
authors: Centre for Biomarker Research and Development, Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, Brisbane, QLD 4072, Australia.
authors: Sakandar
authors: S
authors: Rauf
authors: Centre for Biomarker Research and Development, Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, Brisbane, QLD 4072, Australia.
authors: Muhammad J A
authors: MJ
authors: Shiddiky
authors: Centre for Biomarker Research and Development, Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, Brisbane, QLD 4072, Australia; School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD 4072, Australia. Electronic address: m.trau@uq.edu.au.
authors: Matt
authors: M
authors: Trau
mesh_terms: D001483:Base Sequence; D015374:Biosensing Techniques / Q000295:instrumentation*; D001943:Breast Neoplasms / Q000235:genetics*; D045744:Cell Line, Tumor; D018899:CpG Islands; D004247:DNA / Q000032:analysis / Q000235:genetics*; D019175:DNA Methylation; D055664:Electrochemical Techniques; D004867:Equipment Design; D005260:Female; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D022521:Ligase Chain Reaction / Q000295:instrumentation; D008969:Molecular Sequence Data; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013447:Sulfites / Q000737:chemistry
chemical_list: D013447:Sulfites; D004247:DNA; C042345:hydrogen sulfite
keywords: Breast cancer cell line; CpG resolution; DNAzyme; Electrocatalytic reaction; Ligase chain reaction; Locus-specific DNA methylation