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（由适用提供）：随着新的遗传分析方法的开发，将重点上升到高质量的样品制备以及获得大量高分子量和高纯度DNA的能力上。在DNA测序中，第三代技术可提供长达30 kb的长度读取长度，并且需要高质量的孤立DNA。在基因疗法和DNA疫苗中，高结合能力可能极为罕见地分析或对大型染色体重排的功能分析，高分子量输入DNA会减少在感兴趣的区域中发生碎片的机会。在第一阶段，我们创建了一种新型的热塑性二氧化硅底物，其中包含微观折叠的层次分层和称为纳米宾的纳米级二氧化硅薄片。通过最小化的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，清洗和洗脱方案提取大量（10-100倍）高分子量基因组DNA（> 100 kb），这些协议既快速（<45 min），又是廉价的（$ 0.01）。在此II阶段SBIR中，我们将开发纳米界技术，以解决与当前提取方法存在临界局限性的3个领域。首先，我们将通过开发磁性纳米蛋白底物来创建一种用于高MW提取的方法。其次，当前的自动提取系统仅依赖于珠子和色谱柱，并且倾向于将DNA碎片至<50 kb。我们将开发Magnanobind HTP，这是一种96孔格式，用于高通量，高MW DNA提取，与各种现有的自动提取仪器兼容。第三，我们将开发一个微量的纳米蛋白盒，使用<1 µL或<10 ng的临床样品进行小体积提取。最后，我们将使用3种常见的遗传和表观遗传测定法与微粒，自旋柱和降水方法进行比较，从而验证纳米素DNA/RNA提取平台。 Nanobind可能会立即影响DNA提取市场，因为它独特地结合了微粒和旋转柱的速度和易于性，并与苯酚 - 氯仿的性能相结合。