DNA methylation is an epigenetic regulation of gene expression, which has drawn great attention in biomedical research due to its association with various diseases. A robust, inexpensive platform to detect and quantify the methylation status in a specific genomic region is necessary. In this study, an on-chip analytical technique of cytosine methylation with droplets in a microchannel is proposed. Genomic DNA samples are encapsulated into a series of droplets and transported through a detection region, where a stable temperature gradient is created. As the temperature is elevated from 60 °C to 85 °C, the DNA samples denature and the associated fluorescence signals decay, with the relationship being acquired as the melting curve. The droplets serve as discrete reactors for conducting DNA melting curve analysis in the liquid phase, thereby eliminating the need for immobilization of reagents. Due to a high heating rate and greater enhanced thermal stability, this microchip allows larger melting temperature differences for the samples at different percentages of methylated DNA. It has an enhanced discrimination ability and lower volume consumption, compared to the commercial qPCR machine. This chip enables quantification of the methylation levels of the pluripotent stem cell factor Oct-4 in its distal enhancer (DE) region, with a designed probe after bisulfite treatment and asymmetric PCR.

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使用基于液滴的DNA熔解曲线分析进行DNA甲基化测定

DNA甲基化是基因表达的表观遗传调控，由于其与多种疾病相关，因此在生物医学研究中引起了极大的关注。必须有一个强大，便宜的平台来检测和定量特定基因组区域中的甲基化状态。在这项研究中，提出了微通道中胞嘧啶甲基化与液滴的片上分析技术。基因组DNA样品被封装成一系列液滴，并通过检测区域运输，在该区域中产生稳定的温度梯度。当温度从60°C升高到85°C时，DNA样品变性并且相关的荧光信号衰减，并以熔解曲线的形式获取了该关系。液滴充当离散反应器，用于在液相中进行DNA熔解曲线分析，从而不需要固定试剂。由于具有较高的加热速率和更高的热稳定性，因此该微芯片在甲基化DNA的不同百分比下，允许样品的熔解温度差异更大。与商用qPCR机器相比，它具有增强的辨别能力和较低的体积消耗。该芯片可以定量多能干细胞因子的甲基化水平在其远端增强子（DE）区域中的Oct-4，使用经过亚硫酸氢盐处理和不对称PCR的设计探针。