High Integrity and High Yield DNA Extraction Using a Nanostructured Surface

使用纳米结构表面进行高完整性和高产量 DNA 提取

• 批准号: 8646703
• 负责人: Kelvin Liu
• 金额: $ 17.51万
• 依托单位: CIRCULOMICS, INC.
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-05 至 2015-09-24
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8646703
• 关键词: Affect; Area; Benchmarking; Binding; Biological Assay; Buffers; Cell Count; Chloroform; Cytolysis; DNA; Deposition; Devices; Diagnostic tests; Film; Gel; Gold; Heating; Human Cell Line; Lead; Manuals; Measures; Membrane; Methods; Methylation; Microfabrication; Microfluidics; Molecular Biology; Performance; Phase; Phenols; PicoGreen; Positioning Attribute; Precipitation; Preparation; Process; Protocols documentation; Quantum Dots; RNA; Recovery; Research; Robot; Sampling; Scientist; Silicon Dioxide; Small Business Innovation Research Grant; Speed; Structure; Surface; Technology; Temperature; Testing; Time; Tube; Vendor; base; density; foot; gel electrophoresis; inhibitor/antagonist; instrument; magnetic beads; member; nanomaterials; nanoparticle; nanoscale; nanosensors; nanostructured; novel; particle; public health relevance; research study; single molecule; skills; whole blood bacteria

High Integrity and High Yield DNA Extraction Using a Nanostructured Surface DNA/RNA extraction is a critical first step that underpins nearly all of molecular biology. Phenol/chloroform precipitation remains the gold standard when the highest DNA integrity, yield, and purity are needed. However, the significant manual skill and dexterity required makes this method labor intensive, low throughput, and nearly impossible to automate. While commercial kits and automated instruments using silica columns and beads are capable of high yield, these methods generally result in lower DNA integrity (i.e. more sheared, smaller fragments) and lower purity than well performed phenol/chloroform extractions. Existing silica technologies use porous gels, tightly packed columns, and microparticles to increase surface area, leading to reduced DNA integrity due to high shear forces imparted by flow and mixing. We propose a novel silica coated nanomembrane that uses a hierarchical structure of microscale ridges covered by nanoscale chips to create a flat, non-porous substrate with high surface area. This nanomembrane is easily fabricated using an inexpensive thermoplastic material (i.e. pennies per foot) and is capable of binding >20 mg of DNA per cm2. The nanoscale surface topography enables high surface area silica based DNA extraction while eliminating flow and particle based shear forces to facilitate high yield and high purity DNA recovery with exceptional DNA integrity. DNA is bound by simply allowing the sample to contact the membrane surface rather than flow through it. In Aim 1, we will fabricate a thermoplastic nanomembrane with a high density of nanoscale surface topography to create an inexpensive silica substrate with high surface area for DNA extraction. In Aim 2, we will develop an optimized protocol and buffer set for DNA extraction from human cell lines to obtain high DNA integrity, high extraction yield, and high purity. In Aim 3, we will perform DNA extractions using the nanomembrane, commercial spin-columns, magnetic beads, and phenol/chloroform to benchmark the optimized nanomembrane against existing methods. Through this project, we will develop a DNA extraction nanomembrane that combines the convenience of spin columns with the performance of phenol/chloroform.

使用纳米结构表面的高完整性和高产量DNA提取 DNA/RNA提取是支撑几乎所有分子生物学的关键第一步。苯酚/氯仿 当需要最高的DNA完整性、产率和纯度时，沉淀仍然是金标准。然而，在这方面， 所需的大量手工技能和灵巧性使得该方法劳动强度大、产量低， 几乎不可能实现自动化。虽然商业试剂盒和使用硅胶柱和硅胶柱的自动化仪器 珠能够高产率，但是这些方法通常导致较低的DNA完整性（即更多的剪切， 更小的片段）和比良好进行的苯酚/氯仿提取更低的纯度。现有二氧化硅 技术使用多孔凝胶、紧密填充的柱和微粒来增加表面积， 由于流动和混合产生的高剪切力而降低DNA完整性。我们提出了一种新的二氧化硅涂层 这种纳米膜使用由纳米级芯片覆盖的微米级脊的分层结构来创建一个 具有高表面积的平坦无孔基材。这种纳米膜很容易使用 便宜的热塑性材料（即每英尺几美分），并且能够结合>20 mg DNA/cm 2。 纳米级表面形貌使得能够进行基于高表面积二氧化硅的DNA提取，同时消除了 基于流动和颗粒的剪切力，以促进高产量和高纯度的DNA回收， 完整通过简单地让样品接触膜表面而不是流动来结合DNA 在目标1中，我们将制备具有高密度纳米级表面的热塑性纳米膜 在一个实施例中，该方法包括使用一种新的表面形貌来创建具有用于DNA提取的高表面积的廉价二氧化硅基底。在目标2中， 将开发一种优化的方案和缓冲液组，用于从人类细胞系中提取DNA，以获得高水平的DNA 完整、提取率高、纯度高。在目标3中，我们将使用 纳米膜，商业旋转柱，磁珠，和苯酚/氯仿，以基准 优化的纳米膜对现有的方法。通过这个项目，我们将开发一种DNA提取方法， 纳米膜结合了旋转柱的便利性和苯酚/氯仿的性能。