磁珠负载的DNA三维探针及其在数字PCR循环肿瘤DNA检测中的应用

Circulating tumor DNA (ctDNA), resulting from tumor cell apoptosis and/or necrosis, carries cancer-related gene information and is considered as an important biomarker for the development of “liquid biopsy”. The sensitive analysis of ctDNA still remains challenging because the level of ctDNA is usually very low, and also, it is very unstable for the biological degradation. Digital PCR (dPCR), based on high-throughput and single-molecule PCR amplification, is showing great potential in ctDNA analysis, however, the sensitivity of dPCR is still limited by the specificity of ctDNA enrichment and the PCR amplification efficiency of single DNA fragment. In this work, in order to develop a high-sensitivity and high-specificity ctDNA analysis strategy for the quantitative and qualitative analysis of ctDNA for different cancer samples, we will firstly design and construct a novel multi-dimension DNA probe for specifically capture of ctDNA, using DNA origami technology, and then, we will research its self-assembly on magnetic nano-beads (MNB), and the following application in dPCR analysis based on the successful PCR amplification on the surface of single MNB. Our research will be focused on two parts: firstly, we will research the construction of 3-D DNA probe and the self-assembly on the surface of MNB, to overcome the key problem of the optimization of DNA interfacial configuration, and thus promote the specificity and hybridization efficiency; Secondly, we will study the sample dispersion in dPCR systems under the assistance of MNB, and also the following PCR amplification on the surface of MNB, to develop a theoretical model of PCR on the surface of single MNB, overcoming the key challenge of the PCR amplification of single DNA molecule in a very confined space. The result of our project will hopefully provide reliable analysis methods for the application of ctDNA in cancer diagnosis, treatment and personalized medicine, moreover, we will provide valuable experimental and technical basis for the research of cancer.

循环肿瘤DNA（ctDNA）是来自肿瘤细胞基因组的DNA片段，携带癌症相关特征基因，是“液体活检”的重要发展方向，其含量低、易降解，给检测方法提出挑战，数字PCR基于高通量单分子扩增实现核酸定量，在ctDNA检测方面具有巨大潜力，但ctDNA特异性捕获及单分子扩增效率等问题，仍限制dPCR灵敏度的提高。本项目主要目标是利用DNA折纸技术，设计磁珠负载DNA三维探针，并与dPCR技术结合，实现磁珠表面DNA的PCR单分子扩增，构建高特异性和高灵敏度的ctDNA检测方法，用于多种癌症样品分析。重点研究三维DNA探针构建以及纳米磁珠自组装，解决磁珠表面分子探针构型和数量调控问题；研究微单元样品分散和PCR扩增过程，建立纳米磁珠辅助的PCR扩增理论模型，解决微单元中单分子有效扩增难题。为ctDNA在癌症诊疗和个性化用药方面的应用，提供可靠的分析方法，为癌症的研究提供有力的实验基础和技术依据。

设计不同的二维DNA纳米探针结构，来提高纳米传感界面上的化学稳定性和生物相容性。利用二维DNA纳米结构构建多元、协同的纳米传感界面，并通过调控纳米界面来实现目标DNA分子的高特异性捕获与识别，本文构建了三种电化学DNA生物传感器.多嵌段DNA探针由于开发了多靶点生物传感器和提高了其与目标杂交的特异性/灵敏性而引起了广泛的科学关注。但是多嵌段DNA探针的开发，高度依赖有机连接基团或纳米材料的有机化学合成，因此，限制了它们与目标结合的生物相容性和实用性。在这项工作中，我们开发了基于三嵌段DNA捕获探针的无标记组装策略，该探针是由两个DNA探针与其固有的多腺嘌呤连接起来构成（探针-PolyA-探针，PAP）。中间的polyA片段与金电极表面有很好的结合作用，这使得我们的生物传感器具有极好的重现性、稳定性和再生性。.低浓度水平基因的准确可靠检测是精准医疗面临的一个重要挑战，低浓度水平DNA标准物质是基因检测过程量值溯源的重要途径，可以有效保障检测结果的可靠性。该工作对低浓度水平的短链基因标准物质的定值方法进行了研究，选取TP53基因的一段300 bp长度的序列作为标准物质的目标序列，优化引物探针设计，通过考察PCR扩增的效率可以达到98.6%, 还对数字PCR方法定值的准确性、重复性和重现性进行了研究，最后采用数字PCR方法多家单位联合定值的方法确定了标准物质的终浓度为(620±9.18) copies /µL (k=2), 实现了对低浓度水平短链DNA标准物质的定值。

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