DNA extraction using a tetramethyl orthosilicate-grafted photopolymerized monolithic solid phase.
A novel high-capacity, high-efficiency DNA extraction method is described using a photopolymerized silica-based monolithic column in a fused-silica capillary. Development involved investigation of the composition of the sol-gel monomer, fabrication conditions, and surface modifications in order to optimize the binding capacity. Extraction capacity and efficiency with the 3-(trimethoxysilyl)propyl methacrylate (TMSPM) monolith formulations fabricated in capillaries were investigated using a simple three-step procedure consisting of sample loading, washing of the solid phase, and elution of the DNA using a low ionic strength Tris buffer at pH 8. Once the TMSPM monomer concentration was optimized to yield a monolith with maximum test stability (robustness) and minimum back pressure, the monolith surface was modified by the grafting of tetramethyl orthosilicate (TMOS) for increased DNA binding capacity. After the examination of a variety of TMOS concentrations, 85% v/v TMOS was found to be optimal for DNA extraction without any obvious changes to the monolith structure. The reduction of time allowed for TMSPM hydrolysis prior to UV polymerization from 20 to 5 min led to a lower back pressure of the monolith, enabling better TMOS derivatization and therefore higher binding capacity. Minimal buffer volume (as low as 1 muL) was required to elute DNA from the solid phase, providing a DNA concentrating effect potentially important for downstream processes. While experimentation employed monolithic columns that were 12 cm in length, reduction of the length to 2 cm still allowed for a DNA binding capacity of at least 100 ng of prepurified human genomic DNA and extraction efficiencies greater than 85%. Extraction of low sample volumes (submicroliter) of human whole blood were successfully performed, with extraction efficiencies from the 2-cm monolithic column higher than those obtained from a commercial DNA extraction kit. These results position this novel matrix as an attractive alternative for solid-phase extraction of DNA and other biologically active molecules in microscale devices.
Wen
Jian
J
Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia 22904, USA.
Guillo
Christelle
C
Ferrance
Jerome P
JP
Landers
James P
JP
D001769:Blood; D002845:Chromatography / Q000295:instrumentation / Q000379:methods*; D004247:DNA / Q000302:isolation & purification*; D004867:Equipment Design; D015894:Genome, Human; D006801:Humans; D008722:Methods; D008832:Microchemistry / Q000295:instrumentation*; D017646:Organosilicon Compounds; D011108:Polymers; D012822:Silicon Dioxide
D017646:Organosilicon Compounds; D011108:Polymers; C500739:tetramethyl orthosilicate; D012822:Silicon Dioxide; D004247:DNA