Highly specific, amplification-free, single-molecule counting of rare methylated DNA cancer biomarkers

项目总结 该提案的最终愿景是为高度特定、快速和健壮的 用于诊断和研究的癌症相关DNA甲基化生物标志物的检测。表观遗传 众所周知，改变是癌症中一种关键的调节模式。尤其是DNA甲基化 已知在癌症中存在几十年的失调，并且DNA甲基化的特定位点被寻找为 血液、粪便和其他样本中癌症的非侵入性生物标志物。然而，取得进展的一个主要障碍是 一直缺乏高度敏感、特异和定量的方法来检测DNA甲基化 DNA中的特定位置。几乎所有现有的方法都需要对DNA进行亚硫酸氢盐处理才能转化为 未甲基化的胞嘧啶为尿嘧啶(保留5-甲基胞嘧啶，5mC，完整)，然后进行DNA测序或 定量聚合酶链式反应。然而，亚硫酸氢盐处理是刺激性的，并损害DNA；获得高效率的 转换通常与80%的DNA降解成低扩增片段有关。这, 当与基于聚合酶链式反应扩增的方法相结合时，在检测的灵敏度方面引入了限制 特定部位的甲基化DNA。此外，亚硫酸氢盐处理的DNA的序列复杂性降低- 从本质上讲，将四个字母的遗传密码减少为三个字母的遗传密码--增加了虚假的风险 在基于定量聚合酶链式反应的方法中，扩增和降低了特异性。我们在这里提出了一种方法 DNA甲基化检测和量化在概念上很简单，但利用了复杂的 以及单分子成像科学的优雅进步。该方法基于使用总内部 反射荧光显微镜检测序列特异性的重复结合和释放 荧光标记的探针将目标DNA分子固定在玻片表面。跟随 亚硫酸氢盐转化，这种重复探测(即指纹)单分子的独特方法可以 区分甲基化和未甲基化的靶基因具有极高的特异性(&gt；99.999)和 单分子水平，能够计数每个特定的生物标记分子，同时避免 用聚合酶链式反应检测到假性引爆。与已建立的基于聚合酶链式反应的方法相比，不需要对 这种方法需要分析物DNA，这有望提高对高度碎片的灵敏度 输入DNA。为了验证这种方法并评估其应用于DNA基因座的潜力， 癌症诊断，我们建议开发和基准直接的单分子DNA甲基化分析 使用带有寡核苷酸探针的动态指纹技术来检测在人类中常见的高甲基化 结直肠癌，使用合成样本和患者血液样本。通过开展这项工作，我们将 最大限度地提高成功开发敏感、特异、聚合酶链式反应的变革性新方法的可能性 特定CpG基因座上与癌症相关的DNA甲基化的无扩增定量。

PROJECT SUMMARY The ultimate vision of this proposal is to develop a technology platform for the highly specific, rapid, and robust detection of cancer-associated DNA methylation biomarkers for diagnostics and research. Epigenetic alterations are known to be a crucial mode of regulation in cancer. DNA methylation in particular has been known to be dysregulated in cancer for decades and specific loci of DNA methylation have been sought as non-invasive biomarkers for cancer in blood, stool and other samples. However, a major barrier to progress has been the lack of highly sensitive, specific, and quantitative methods for detecting DNA methylation at specific sites in DNA. Nearly all established methods require bisulfite treatment of the DNA to convert unmethylated cytosines to uracil (leaving 5-methylcytosine, 5mC, intact), followed by DNA sequencing or quantitative PCR. However, bisulfite treatment is harsh and damages the DNA; obtaining a high efficiency of conversion is typically associated with degradation of >80% of the DNA into poorly amplified fragments. This, when combined with PCR-amplification-based methods, introduces limitations in the sensitivity of detecting methylated DNA at specific sites. Furthermore, the reduced sequence complexity of bisulfite-treated DNA— essentially reducing the four-letter genetic code to a three-letter one—increases the risk of spurious amplification and reduced specificity in quantitative PCR-based approaches. We here propose an approach to DNA methylation detection and quantification that is conceptually simple, yet takes advantage of sophisticated and elegant advances in single-molecule imaging science. The approach is based on using total internal reflection fluorescence microscopy to detect the repeated binding and release of sequence-specific fluorescently tagged probes to immobilized target DNA molecules on the surface of a glass slide. Following bisulfite conversion, this unique approach of repetitive probing (i.e., fingerprinting) of single molecules can distinguish between methylated and unmethylated target genes with exquisite specificity (>99.999%) and at the single-molecule level, enabling counting each specific biomarker molecule while avoiding the problem of spurious priming seen with PCR. In contrast to established PCR-based methods, no enzymatic manipulation of the analyte DNA is required with this approach, which is expected to improve sensitivity for highly fragmented input DNA. In order to validate this approach and assess its potential for application to DNA loci important in cancer diagnostics, we propose to develop and benchmark a direct single-molecule DNA methylation assay using kinetic fingerprinting with oligonucleotide probes against loci that are commonly hypermethylated in colorectal cancer, using both synthetic samples and patient blood specimens. By pursuing this work, we will maximize the likelihood of success in developing a transformative new approach for sensitive, specific, PCR amplification-free quantification of cancer-relevant DNA methylation at specific CpG loci.