Multiplexed Detection of Cell Free DNA Biomarkers for Cancer

描述(申请人提供)：循环中的无细胞DNA已被发现自由存在于人体体液中，如血液、尿液和粪便。这些稀有的核酸包含一群遗传和表观遗传的生物标记物，可以用来揭示隐藏的病理。在癌症中，无细胞DNA可以通过检测与肿瘤相关的分子变化(如点突变和DNA甲基化)来确定远程肿瘤的状态，在非侵入性监测肿瘤动力学、治疗反应和疾病进展方面具有很大的前景。由于无细胞DNA的生理浓度很低，因此突变等位基因特异性扩增(MASA)和甲基化特异性聚合酶链式反应(MSP)是目前主流的DNA分析方法。不幸的是，这些方法的吞吐量和多路复用度一直受到限制。鉴于癌症之间在分子变化上存在显著的异质性，需要对一组生物标志物进行分析，以确定组织类型和恶性转化。然而，多重聚合酶链式反应的努力一直受到错误引发和彩色荧光染料供应有限等问题的阻碍。对每个样品使用大量单独的PCR是昂贵和有问题的，因为它需要大量的DNA，而DNA是无细胞DNA分析的限制因素。我们寻求开发一种微流控单分子检测平台，以满足对无细胞DNA生物标记物的多路分析和高通量分析的需求。我们的平台利用单分子光谱学的高灵敏度，实现了对低浓度DNA的直接分析，而不依赖于聚合酶链式反应。它的目标是通过使用分子编码方法来生成生物标记物特定的氟代码，然后通过多色单分子光谱学进行解码，从而在一次测量中实现对e 48生物标记物的高度多元化检测。此外，该平台还集成了用于并行目标浓度和阵列检测的微流控设备，便于在一个芯片上高通量测量50个样品。从卵巢癌、肺癌和结肠癌患者的血清、痰和粪便样本中检测遗传和表观遗传癌症生物标记物，包括点突变和启动子DNA甲基化，就证明了该平台的能力。 公共卫生相关性：该项目的目标是开发一个微流控单分子检测平台，用于分析体液中的无细胞DNA生物标记物。这一新平台将允许对一个DNA样本中的e 48个生物标记物进行多路检测，并对每个芯片上的50个样本进行并行测量，这是目前主流的、基于聚合酶链式反应的无细胞DNA检测方法所无法实现的。

DESCRIPTION (provided by applicant): Circulating cell-free DNA have been found to freely exist in human body fluids such as blood, urine, and stool. These rare nucleic acids contain a bevy of genetic and epigenetic biomarkers that can be used to reveal hidden pathologies. In cancer, cell-free DNA can be used to determine the status of remote tumors through detecting tumor-associated molecular alterations, such as point mutations and DNA methylation, with great promise for non-invasive monitoring of tumor dynamics, therapeutic response, and disease progression. Since cell-free DNA are present at very low physiological concentrations, PCR-based methodologies, such as mutation allele specific amplification (MASA) and methylation-specific PCR (MSP), are current mainstream methods for their analysis. Unfortunately, the throughput and multiplexing of these methods have been limited. Given that substantial heterogeneity in molecular alterations exists among cancers, analysis of a panel of biomarkers is needed to determine the tissue type and malignant transformation. However, efforts to multiplex PCR have been hampered by issues including mispriming and limited availability of colored florescent dyes. Employing a large number of separate PCRs for each sample is costly and problematic as it requires large amounts of DNA where DNA is the limiting factor for cell-free DNA analysis. We seek to develop a microfluidic single-molecule detection platform to address the unmet need for multiplexed and high-throughput analysis of cell-free DNA biomarkers. Our platform takes advantage of the high sensitivity of single-molecule spectroscopy to enable direct analysis of low-concentration DNA without reliance on PCR. It aims to achieve highly multiplexed detection of e 48 biomarkers in a single measurement via the use of a molecular coding approach to generate biomarker-specific fluoro-codes that are subsequently decoded by multi-color, single-molecule spectroscopy. In addition, the platform incorporates a microfluidic device for parallel target concentration and arrayed detection, facilitating high-throughput measurements of 50 samples on a chip. The capability of the platform is exemplified by detection of both genetic and epigenetic cancer biomarkers, including point mutations and promoter DNA methylation in serum, sputum and stool samples, collected from patients with ovarian, lung and colon cancer. PUBLIC HEALTH RELEVANCE: The goal of the project is to develop a microfluidic single-molecule detection platform for analysis of cell-free DNA biomarkers in body fluids. This new platform will allow multiplexed detection of e 48 biomarkers in a DNA sample and parallel measurements of 50 samples per chip, unattainable by the current mainstream, PCR-based methods for cell-free DNA detection.