title: Droplet Array Platform for High-Resolution Melt Analysis of DNA Methylation Density.
abstract: High-resolution melting (HRM) has garnered significant interest as an analytical technique for a number of applications, including DNA methylation detection, due to its inherent sensitivity and robustness. In this study, we describe a miniaturized assay platform for quantitative methylation density analysis using a microfluidic droplet array cartridge. We demonstrate that the DNA methylation level of the RASSF1A promoter can be directly analyzed using HRM. PCR products were generated by amplifying bisulfite-treated DNA with varying CpG densities using CpG island-flanking primer sets. Subsequent HRM analysis on the miniaturized droplet platform shows distinct melting curve profiles associated with methylation levels, which was verified using a conventional benchtop PCR-HRM system. The characteristic melting temperature (Tm) of the PCR products was used to directly quantify the respective levels of DNA methylation density. Our approach provides a key advantage over current gold standard methods such as methylation-specific PCR (MSP), which are incapable of providing specific information regarding the overall methylation density of the target genes. The miniaturized platform establishes a practical approach to methylation density profiling from multiple DNA samples with a potential application in point-of-care diagnostics.
authors.affiliation: Department of Biomedical Engineering, The Johns Hopkins University, Baltimore, MD, USA.
authors.forename: Pornpat
authors.initials: P
authors.lastname: Athamanolap
authors.affiliation: Department of Biomedical Engineering, The Johns Hopkins University, Baltimore, MD, USA.
authors.forename: Dong Jin
authors.initials: DJ
authors.lastname: Shin
authors.affiliation: Department of Biomedical Engineering, The Johns Hopkins University, Baltimore, MD, USA Department of Mechanical Engineering, The Johns Hopkins University, Baltimore, MD, USA thwang@jhu.edu.
authors.forename: Tza-Huei
authors.initials: TH
authors.lastname: Wang
mesh_terms: D057205:Automation, Laboratory; D002138:Calibration; D018899:CpG Islands; D019175:DNA Methylation; D004274:DNA, Recombinant / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007202:Indicators and Reagents; D046228:Microarray Analysis; D046210:Microfluidic Analytical Techniques; D008904:Miniaturization; D009691:Nucleic Acid Denaturation; D000067716:Point-of-Care Testing; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D000075082:Proof of Concept Study; D015203:Reproducibility of Results; D013447:Sulfites; D044366:Transition Temperature; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D004274:DNA, Recombinant; D007202:Indicators and Reagents; C411762:RASSF1 protein, human; D013447:Sulfites; D025521:Tumor Suppressor Proteins; C042345:hydrogen sulfite
keywords: DNA methylation detection; melt curve analysis; miniaturized system