This work presents a digital microfluidic platform called HYPER-Melt (high-density profiling and enumeration by melt) for highly parallelized copy-by-copy DNA molecular profiling. HYPER-Melt provides a facile means of detecting and assessing sequence variations of thousands of individual DNA molecules through digitization in a nanowell microchip array, allowing amplification and interrogation of individual template molecules by detecting HRM fluorescence changes due to sequence-dependent denaturation. As a model application, HYPER-Melt is used here for the detection and assessment of intermolecular heterogeneity of DNA methylation within the promoters of classical tumor suppressor genes. The capabilities of this platform are validated through serial dilutions of mixed epialleles, with demonstrated detection limits as low as 1 methylated variant in 2 million unmethylated templates (0.00005%) of a classic tumor suppressor gene, CDKN2A (p14^ARF). The clinical potential of the platform is demonstrated using a digital assay for NDRG4, a tumor suppressor gene that is commonly methylated in colorectal cancer, in liquid biopsies of healthy and colorectal cancer patients. Overall, the platform provides the depth of information, simplicity of use, and single-molecule sensitivity necessary for rapid assessment of intermolecular variation contributing to genetic and epigenetic heterogeneity for challenging applications in embryogenesis, carcinogenesis, and rare biomarker detection.

中文翻译：

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通过微流数字熔体轻松进行分子异质性分析。

这项工作提出了一种称为HYPER-Melt（高密度分析和熔体枚举）的数字微流控平台，用于高度并行的逐拷贝DNA分子分析。HYPER-Melt提供了一种简便的方法，可以通过在纳米孔微芯片阵列中进行数字化来检测和评估成千上万个单个DNA分子的序列变异，从而可以通过检测由于序列依赖性变性导致的HRM荧光变化来扩增和询问单个模板分子。作为模型应用，HYPER-Melt在这里用于检测和评估经典肿瘤抑制基因启动子内DNA甲基化的分子间异质性。该平台的功能通过混合稀释的等位基因的系列稀释得到验证，CDKN2A（p14 ^ARF）。该平台的临床潜力在健康和大肠癌患者的液体活检中使用NDRG4（一种在大肠癌中通常被甲基化的抑癌基因）的数字化验方法得到了证明。总体而言，该平台可提供信息深度，易用性以及快速评估分子间变异所必需的信息，这些分子间变异有助于遗传和表观遗传异质性，可在胚胎发生，致癌作用和稀有生物标志物检测中具有挑战性的应用。