Multiplexed Detection of Cell Free DNA Biomarkers for Cancer

描述（由申请人提供）：循环无细胞DNA已被发现自由存在于人体体液中，如血液、尿液和粪便。这些罕见的核酸含有大量的遗传和表观遗传生物标志物，可用于揭示隐藏的病理。在癌症中，无细胞DNA可以通过检测肿瘤相关的分子改变（如点突变和DNA甲基化）来确定远处肿瘤的状态，对于肿瘤动力学、治疗反应和疾病进展的无创监测具有很大的前景。由于无细胞DNA以非常低的生理浓度存在，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)，是目前分析它们的主流方法。不幸的是，这些方法的吞吐量和多路复用是有限的。考虑到癌症分子变化的异质性，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，多重聚合酶链反应的努力受到了包括引物错误和有色荧光染料有限可用性等问题的阻碍。对每个样品使用大量分离的pcr是昂贵的，并且存在问题，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，以解决对无细胞DNA生物标志物的多路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的DNA，而不依赖于PCR。它旨在通过使用分子编码方法在单次测量中实现对48种生物标志物的高度多路检测，以生成生物标志物特异性荧光编码，随后通过多色单分子光谱解码。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装置，便于在芯片上对50个样品进行高通量测量。通过检测卵巢癌、肺癌和结肠癌患者的血清、痰和粪便样本中的基因和表观遗传癌症生物标志物，包括点突变和启动子DNA甲基化，该平台的能力得到了证明。

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.