Nanobind Hierarchical Silica Lamella for High Molecular Weight DNA Extraction

用于高分子量 DNA 提取的 Nanobind 分层二氧化硅片层

• 批准号: 9150684
• 负责人: Kelvin Liu
• 金额: $ 74.86万
• 依托单位: CIRCULOMICS, INC.
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-25 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9150684
• 关键词: Address; Area; Binding; Biological Assay; Biological Markers; Biological Sciences; Biopsy Specimen; Biotechnology; Body Fluids; Buffers; Cells; Chloroform; Chromosomal Rearrangement; Clinical; DNA; DNA Binding; DNA Sequence; DNA Vaccines; Data Quality; Detection; Diagnostic; Epigenetic Process; Film; Fluorescence; Generations; Genetic; Genetic Research; Genomic DNA; Goals; Gold; Health; Heating; Length; Magnetism; Marketing; Membrane; Methods; Methylation; MicroRNAs; Microfluidics; Molecular; Molecular Conformation; Molecular Weight; Mutation Detection; Nucleic Acids; Output; Performance; Phase; Phenols; Plasmids; Precipitation; Preparation; Process; Protocols documentation; RNA; Reading; Robot; Sampling; Scientist; Silicon Dioxide; Small Business Innovation Research Grant; Speed; Surface; System; Systems Development; Technology; Tissues; Vendor; Veterans; Work; clinical application; cost; experience; gene therapy; genetic analysis; improved; instrument; interest; magnetic beads; nanomaterials; nanoscale; novel; particle; polyolefin; sequencing platform; single molecule; tool

 DESCRIPTION (provided by applicant): As new genetic analysis methods are developed, rising emphasis is being placed on high quality sample preparation and the ability to obtain large quantities of high molecular weight and high purity DNA. In DNA sequencing, 3rd generation technologies offer long read lengths up to 30 kb and require high quality isolated DNA. In gene therapy and DNA vaccines, high binding capacity could be extremely useful in isolating industrial quantities of long plasmids. In genetic analysis of extremely rare sequences or functional analysis of large chromosomal rearrangements, high molecular weight input DNA reduces the chance that fragmentation will occur in a region of interest. In Phase I, we created a novel thermoplastic, silica substrate, containing a hierarchical layering of microscale folds and nanoscale silica lamella called Nanobind. Through the combined effects of minimized shear force exposure and high surface area, we are able to extract vast amounts (10- 100X more) of high molecular weight genomic DNA (>100 kb) using a simple bind, wash, and elute protocol that is both fast (<45 min) and inexpensive ($0.01 per substrate). In this Phase II SBIR, we will develop Nanobind technologies to address 3 areas where critical limitations exist with current extraction methods. First, we will create a facile method for high MW extraction by developing a magnetic Nanobind substrate. Second, current automated extraction systems rely exclusively on beads and columns and tend to fragment DNA to <50 kb. We will develop MagNanobind HTP, a 96-well format for high-throughput, high MW DNA extraction that is compatible with a variety of existing automated extraction instruments. Third, we will develop a microvolume Nanobind cartridge to perform small volume extractions using clinical samples <1 µL or <10 ng. Finally, we will validate the Nanobind DNA/RNA extraction platform by comparing against microparticle, spin column, and precipitation methods using 3 common genetic and epigenetic assays. Nanobind could immediately impact the DNA extraction market as it uniquely combines the speed and ease of microparticles and spin columns with performance of phenol-chloroform.

 描述(申请人提供)：随着新的遗传分析方法的发展，人们越来越重视高质量的样品制备和获得大量高相对分子质量和高纯度DNA的能力。在DNA测序方面，第三代技术提供高达30kb的长阅读长度，并需要高质量的分离DNA。在基因治疗和DNA疫苗中，高结合能力在分离工业化数量的长质粒方面可能非常有用。在极其罕见的序列的遗传分析或大型染色体重排的功能分析中，输入高分子量的DNA可以降低在感兴趣区域发生片段化的可能性。在第一阶段，我们创建了一种新型的热塑性二氧化硅衬底，包含微米级折叠和纳米级二氧化硅薄片的分层，称为Nanobind。通过最小剪切力暴露和高表面积的综合作用，我们能够使用简单的结合、洗涤和洗脱方案提取大量(10-100倍以上)的高分子量基因组DNA(&gt；100kb)，既快速(&lt；45分钟)又便宜(每个底物0.01美元)。在这个第二阶段的SBIR中，我们将开发纳米结合技术，以解决当前提取方法存在严重限制的三个领域。首先，我们将通过开发磁性纳米结合剂衬底来创建一种简便的高MW提取方法。其次，目前的自动提取系统完全依赖于珠子和柱子，往往会将DNA片段切成50kb。我们将开发MagNanobind HTP，这是一种96孔格式的高通量、高分子量DNA提取，与现有的各种自动提取仪器兼容。第三，我们将开发一种微型纳米结合色谱柱，用于使用临床样本&lt；1或&lt；10 ng进行小容量提取。最后，我们将使用3种常见的遗传和表观遗传学分析方法，通过与微粒法、自旋柱法和沉淀法的比较来验证Nanobind DNA/RNA提取平台的有效性。Nanobind可以立即影响DNA提取市场，因为它独特地将微粒和旋转柱的速度和易用性与苯酚-氯仿的性能结合在一起。