Department of Biomedical Engineering, Johns Hopkins School of Medicine, Baltimore, MD 21218, USA.
Lab Chip. 2019 Jan 29;19(3):444-451. doi: 10.1039/c8lc01189c.
Liquid biopsies contain a treasure of genetic and epigenetic biomarkers that contain information for the detection and monitoring of human disease. DNA methylation is an epigenetic modification that is critical to determining cellular phenotype and often becomes altered in many disease states. In cancer, aberrant DNA methylation contributes to carcinogenesis and can profoundly affect tumor evolution, metastatic potential, and resistance to therapeutic intervention. However, current technologies are not well-suited for quantitative assessment of DNA methylation heterogeneity, especially in challenging samples such as liquid biopsies with low DNA input and high background. We present a multilayer microfluidic device for quantitative analysis of DNA methylation by digital PCR and high resolution melt (HRM). The multilayer design facilitates high-density array digitization aimed at maximizing sample loading efficiency. The platform achieves highly parallelized digital PCR-HRM-based discrimination of rare heterogeneous DNA methylation as low as 0.0001% methylated/unmethylated molecules of a classic tumor suppressor gene, CDKN2A (p14ARF).
液体活检包含了大量的遗传和表观遗传生物标志物，这些标志物包含了人类疾病检测和监测的信息。DNA 甲基化是一种表观遗传修饰，对确定细胞表型至关重要，并且在许多疾病状态下经常发生改变。在癌症中，异常的 DNA 甲基化导致了致癌作用，并可能深刻地影响肿瘤的进化、转移潜力和对治疗干预的抵抗。然而，目前的技术并不适合定量评估 DNA 甲基化异质性，特别是在具有挑战性的样本中，如液体活检，其 DNA 输入量低，背景高。我们提出了一种用于通过数字 PCR 和高分辨率熔解（HRM）定量分析 DNA 甲基化的多层微流控装置。多层设计有利于高密度阵列数字化，旨在最大限度地提高样品加载效率。该平台实现了基于高度并行的数字 PCR-HRM 对罕见异质 DNA 甲基化的区分，其灵敏度低至 0.0001%甲基化/未甲基化的经典肿瘤抑制基因 CDKN2A（p14ARF）的分子。
