Chitosan as a polymer for pH-induced DNA capture in a totally aqueous system.
A novel DNA solid-phase extraction protocol based on the pH-dependent charge of chitosan was developed specifically for low-volume DNA extraction on microchips. The method uses chitosan-coated beads to extract DNA at pH 5 and release it from the chitosan at pH 9. DNA extraction efficiency as high as 92% could be attained, even from complex samples such as human blood containing significant amounts of protein. Using this method, PCR inhibitors that are typically used in DNA extraction procedures (e.g., chaotropic salts, 2-propanol) can be avoided, making the method more conducive to downstream sample processing using PCR. A high-density multichannel microchip device was then fabricated and the microchannels coated with chitosan for DNA extraction in an open channel configuration without the need for an additional stationary phase. This design provided a relatively high surface area-to-volume ratio for extraction, while retaining the low flow resistance commensurate with open channels. With a flow rate of approximately 1 microL/min during the extraction, the total extraction time was less than 10 min, with most of the DNA recovered in the first 2 microL of elution buffer. Using the microchip device, extraction efficiencies for lambda-phage DNA and human genomic DNA were as high as 67 and 63%, respectively. Human genomic DNA from whole blood samples could be extracted in 10 min with an extraction efficiency of 75 +/- 4% (n = 3), and the purified DNA was suitable for PCR amplification of a fragment of the gelsolin gene. The combination of an entirely aqueous DNA extraction method with a high-density, low-flow resistance microchannel pattern sets the stage for future integration into microfluidic genomic analysis devices.
Cao
Weidong
W
Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, USA.
Easley
Christopher J
CJ
Ferrance
Jerome P
JP
Landers
James P
JP
D048271:Chitosan / Q000737:chemistry*; D004247:DNA / Q000737:chemistry* / Q000302:isolation & purification; D015894:Genome, Human; D006801:Humans; D006863:Hydrogen-Ion Concentration; D046208:Microchip Analytical Procedures; D016133:Polymerase Chain Reaction; D012822:Silicon Dioxide / Q000737:chemistry
D012822:Silicon Dioxide; D004247:DNA; D048271:Chitosan