DNA Methylation Analysis Using Solid-State Nanopore Sensors - A Pathway to Early

描述（由申请人提供）：2004年，癌症是全球死亡的主要原因，约占所有死亡人数的13%。越来越明显的是，癌症既是一种基因突变疾病，也是一种被错误引导的表观遗传学疾病。表观遗传改变以DNA甲基化改变的形式发生，这是癌变的早期和经常观察到的事件。有趣的是，来自大多数肿瘤类型的癌症特异性甲基化DNA很容易在体液和活检标本中找到，也以死亡癌细胞脱落的自由漂浮DNA的形式存在。一种能够检测从癌症患者血清中提取的特定基因的异常甲基化模式的技术将具有巨大的临床价值。我们建议使用固态纳米孔传感器来检测人类血清样本中超低浓度的强大癌症生物标志物（特别是DNA甲基化模式）。纳米孔传感器利用电流光谱原理来检测单个DNA分子，其灵敏度可以识别单个分子中细微的结构基序。纳米孔技术也非常适合于基于基因的甲基化分析，能够筛选各种癌症特异性高甲基化标记的小小组。纳米孔传感器可能在早期癌症检测、风险评估、疾病监测、化学预测和患者预后方面发挥重要作用。为了实现我们基于纳米孔的甲基化分析的目标，在本R21中，我们提出了以下具体目标：(i)我们将首先使用市上可用的、完全甲基化的DNA片段探索纳米孔传感器的甲基化检测能力，以及（ii）我们将进行甲基化质粒DNA的甲基化检测和定量。根据目标1和目标2的结果，该技术可以在R01的基础上扩展到临床前和临床样本的分析，特别是检测从前列腺癌患者血清中分离的DNA中的异常甲基化模式。

DESCRIPTION (provided by applicant): Cancer is a leading cause of death worldwide accounting for approximately 13% of all deaths in 2004. It is becoming more and more apparent that cancer is as much a disease of misdirected epigenetics as it is a disease of genetic mutations. Epigenetic alterations occur in the form of DNA methylation changes, an early and frequently observed event in carcinogenesis. Interestingly, cancer-specific methylated DNA from most tumor types is readily available in bodily fluids and biopsy specimens and also exists in the form of free-floating DNA shed by dead cancer cells. A technology capable of detecting aberrant methylation patterns in specific genes extracted from the serum of cancer patients would be of immense clinical value. We propose using solid-state nanopore sensors for the detection of robust cancer biomarkers (specifically DNA methylation patterns) at ultra low concentrations in human serum samples. Nanopore sensors use the principle of electrical current spectroscopy to interrogate individual DNA molecules, with the sensitivity to discern subtle structural motifs in single molecules. Nanopore technology is also well suited for gene based methylation analysis, capable of screening small panels of hypermethylation markers specific to a variety of cancers. Nanopore sensors could potentially play an important role in early cancer detection, risk assessment, disease monitoring, chemoprediction and patient prognosis. In working towards our goal of nanopore based methylation analysis, in this R21 we propose the following specific aims: (i) We will first explore the methylation detection capabilities of nanopore sensors using commercially available, fully methylated DNA fragments, and (ii) We will perform Methylation detection and quantification of methylated plasmid DNA. Depending on the outcomes of Aims 1 and 2, this technique may be extended in a follow on R01 to the analysis of pre-clinical and clinical samples, specifically the detection of aberrant methylation patterns in DNA isolated from the serum of prostate cancer patients. PUBLIC HEALTH RELEVANCE: Direct detection of DNA methylation patterns in cancer genes can be very important in early cancer detection, risk assessment, disease monitoring, and patient prognosis. We propose the use of solid-state nanopore sensors for the detection of these methylation patterns.