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.

利用纳米结构表面提取高完整性和高产率的DNA