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.
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. We propose a novel silica coated nanomembrane containing a high density of nanoscale topography that combines the DNA extraction performance of phenol/chloroform with the convenience of spin columns and magnetic beads. This method will facilitate both research assays and diagnostic tests where high DNA quality is required.
|Zhang, Ye; Zhang, Yi; Burke, Jeffrey M et al. (2016) A Simple Thermoplastic Substrate Containing Hierarchical Silica Lamellae for High-Molecular-Weight DNA Extraction. Adv Mater 28:10630-10636|
|Nguyen, Trung C; Cruz, Miguel A; Carcillo, Joseph A (2015) Thrombocytopenia-Associated Multiple Organ Failure and Acute Kidney Injury. Crit Care Clin 31:661-74|