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.

使用纳米结构表面进行高完整性和高产量 DNA 提取 DNA/RNA 提取是支撑几乎所有分子生物学的关键的第一步。苯酚/氯仿 当需要最高的 DNA 完整性、产量和纯度时，沉淀仍然是黄金标准。然而， 所需的大量手工技能和灵活性使得该方法劳动强度大、通量低，并且 几乎不可能实现自动化。虽然商业套件和自动化仪器使用硅胶柱和 珠子能够获得高产率，但这些方法通常会导致 DNA 完整性降低（即更多的剪切、 与执行良好的苯酚/氯仿萃取相比，片段较小）且纯度较低。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，从而导致 由于流动和混合产生的高剪切力，DNA 完整性降低。我们提出了一种新型二氧化硅涂层 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 具有高表面积的平坦、无孔基材。这种纳米膜很容易使用 廉价的热塑性材料（即每英尺几美分），每平方厘米能够结合 >20 毫克的 DNA。 纳米级表面形貌能够实现基于二氧化硅的高表面积 DNA 提取，同时消除 基于流动和颗粒的剪切力，以促进高产量和高纯度 DNA 回收，并具有出色的 DNA 正直。 DNA 通过简单地让样品接触膜表面而不是流动来结合 通过它。在目标 1 中，我们将制造具有高密度纳米级表面的热塑性纳米膜 形貌以创建具有高表面积的廉价二氧化硅基质，用于 DNA 提取。在目标 2 中，我们 将开发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 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/氯仿来对 针对现有方法优化纳米膜。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。