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

描述(申请人提供)：癌症是全球主要的死亡原因，约占2004年所有死亡人数的13%。越来越明显的是，癌症是一种错误的表观遗传学疾病，也是一种基因突变疾病。表观遗传学改变以DNA甲基化改变的形式发生，这是癌症发生的早期和经常观察到的事件。有趣的是，大多数肿瘤类型的癌症特异性甲基化DNA在体液和活检标本中都很容易获得，也以死亡癌细胞脱落的自由漂浮DNA的形式存在。一项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在超低浓度的人类血清样本中检测强大的癌症生物标记物(特别是DNA甲基化模式)。纳米孔传感器使用电流光谱学的原理来询问单个DNA分子，并具有识别单个分子中微妙结构基序的灵敏度。纳米孔技术也非常适合于基于基因的甲基化分析，能够筛选出针对各种癌症的高甲基化标记的小面板。纳米孔传感器可能在癌症早期检测、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，在这份R21中，我们提出了以下具体目标：(I)我们将首先使用商业上可获得的完全甲基化的DNA片段来探索纳米孔传感器的甲基化检测能力，以及(Ii)我们将对甲基化的质粒DNA进行甲基化检测和量化。根据AIMS 1和2的结果，这项技术可能会在R01的后续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提取的DNA的异常甲基化模式。 公共卫生相关性：直接检测癌症基因中的DNA甲基化模式对于癌症早期检测、风险评估、疾病监测和患者预后非常重要。我们建议使用固态纳米孔传感器来检测这些甲基化模式。

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.