Toward a microchip-based solid-phase extraction method for isolation of nucleic acids.
A silica-based solid-phase extraction system suitable for incorporation into a microchip platform (nu-total analytical system; nu-TAS) would find utility in a variety of genetic analysis protocols, including DNA sequencing. The extraction procedure utilized is based on adsorption of the DNA onto bare silica. The procedure involves three steps: (i) DNA adsorption in the presence of a chaotropic salt, (ii) removal of contaminants with an alcohol/water solution, and (iii) elution of the adsorbed DNA in a small volume of buffer suitable for polymerase chain reaction (PCR) amplification. Multiple approaches for incorporation of this protocol into a microchip were examined with regard to extraction efficiency, reproducibility, stability, and the potential to provide PCR-amplifiable DNA. These included packing microchannels with silica beads only, generating a continuous silica network via sol-gel chemistry, and combinations of these. The optimal approach was found to involve immobilizing silica beads packed into the channel using a sol-gel network. This method allowed for successful extraction and elution of nanogram quantities of DNA in less than 25 min, with the DNA obtained in the elution buffer fraction. Evaluation of the eluted DNA indicated that it was of suitable quality for subsequent amplification by PCR.
Wolfe
Kelley A
KA
Department of Chemistry, University of Virginia, Charlottesville, VA 22904, USA.
Breadmore
Michael C
MC
Ferrance
Jerome P
JP
Power
Mary E
ME
Conroy
John F
JF
Norris
Pamela M
PM
Landers
James P
JP
D004247:DNA / Q000302:isolation & purification*; D019075:Electrophoresis, Capillary / Q000379:methods; D007202:Indicators and Reagents; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D012822:Silicon Dioxide
D007202:Indicators and Reagents; D012822:Silicon Dioxide; D004247:DNA