Multiplexed Detection of Cell Free DNA Biomarkers for Cancer

描述（由申请人提供）：已发现循环无细胞DNA自由存在于人体体液（如血液、尿液和粪便）中。这些罕见的核酸含有一系列遗传和表观遗传生物标志物，可用于揭示隐藏的病理学。在癌症中，无细胞DNA可用于通过检测肿瘤相关的分子改变（如点突变和DNA甲基化）来确定远端肿瘤的状态，这对于肿瘤动力学、治疗反应和疾病进展的非侵入性监测具有很大的希望。由于游离DNA的生理浓度非常低，基于PCR的方法，如突变等位基因特异性扩增（MASA）和甲基化特异性PCR（MSP），是目前主流的分析方法。不幸的是，这些方法的吞吐量和多路复用受到限制。鉴于癌症之间存在分子改变的实质异质性，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PCR的努力受到包括错误引发和有色荧光染料的有限可用性的问题的阻碍。对每个样品采用大量单独的PCR是昂贵的和有问题的，因为它需要大量的DNA，其中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，以解决游离DNA生物标志物的多重和高通量分析的未满足需求。我们的平台利用单分子光谱的高灵敏度，可以直接分析低浓度DNA，而不依赖于PCR。其目的是通过使用分子编码方法生成生物标志物特异性荧光代码，随后通过多色单分子光谱法解码，在单次测量中实现e48生物标志物的高度多重检测。此外，该平台还集成了一个用于平行目标浓度和阵列检测的微流体设备，便于在芯片上进行50个样品的高通量测量。该平台的能力通过检测遗传和表观遗传癌症生物标志物来举例说明，包括从卵巢癌、肺癌和结肠癌患者收集的血清、痰液和粪便样本中的点突变和启动子DNA甲基化。 公共卫生相关性：该项目的目标是开发一种微流控单分子检测平台，用于分析体液中的无细胞DNA生物标志物。这个新平台将允许在DNA样品中多重检测e48生物标志物，并在每个芯片上平行测量50个样品，这是目前主流的基于PCR的无细胞DNA检测方法无法实现的。

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.