DNA Extraction and PCR Amplification Performed in a Single Microfluidic Chamber
Integrated microdevices provide the opportunity to encompass multiple analytical steps on a single device, with the possibility of automating the entire sample analysis process. Current work being developed involves a glass microdevice that has been designed to perform solid phase extraction (SPE) of DNA and IR-PCR (infrared-mediated) amplification in a novel format – with both processes in the same chamber. This provides an inherent advantage over any microchip-based DNA extraction described previously [1], in that all of the sample DNA is used for nanoliter amplification, improving detection limits by 1-2 orders of magnitude. A novel solid phase, chitosan-coated magnetic beads, has been developed that has high DNA recoveries (72% ?6%) using a pH-induced DNA release technique [2], while eliminating the use of high salt solutions and organics (both potent PCR inhibitors). A simple external magnet is used to control the location of the beads in the chamber, removing the need for etching structures (such as weirs or pillars) into the channels, increasing the simplicity of device design and fabrication. While previous SPE phases have been composed of materials incompatible with PCR [1] (due to high protein adsorption) the chitosan coating does not affect the efficiency of PCR, allowing the beads to remain in the very same chamber used for PCR.
1. Legendre, L.A., Bienvenue, J.M., Roper, M.R., Ferrance, J.P., Landers, J.P. Anal Chem, 2006, 78, 1444.
2. Cao, W., Easley, C.J., Ferrance, J.P., Landers, J.P. Anal Chem, 2006, 78, 7222-7228.
1. Legendre, L.A., Bienvenue, J.M., Roper, M.R., Ferrance, J.P., Landers, J.P. Anal Chem, 2006, 78, 1444.
2. Cao, W., Easley, C.J., Ferrance, J.P., Landers, J.P. Anal Chem, 2006, 78, 7222-7228.
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