Droplet Array Platform for High-Resolution Melt Analysis of DNA Methylation Density. (June 2014)

• Record Type: Journal Article
• Authors: Athamanolap, Pornpat; Shin, Dong Jin; Wang, Tza-Huei
• Is Part Of: Journal of laboratory automation. Volume 19:Number 3(2014:Jun.)
• Page Start: 304
• Page End: 312
• Publication Date: 2014-06
• Subjects: DNA methylation detection -- melt curve analysis -- miniaturized system; Medical laboratory technology -- Periodicals; Laboratories -- Equipment and supplies -- Periodicals; Diagnosis, Laboratory -- Periodicals; 616.075
• DOI: 10.1177/2211068213507923
• Languages: English
• ISSNs: 2211-0682

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.