Evaluation of silica resins for direct and efficient extraction of DNA from complex biological matrices in a miniaturized format.
For DNA purification to be functionally integrated into the microchip for high-throughput DNA analysis, a miniaturized purification process must be developed that can be easily adapted to the microchip format. In this study, we evaluate the effectiveness of a variety of silica resins for miniaturized DNA purification and gauge the potential usefulness for on-chip solid-phase extraction. A micro-solid-phase extraction (muSPE) device containing only nanograms of silica resin is shown to be effective for the adsorption and desorption of DNA in the picogram-nanogram mass range. Fluorescence spectroscopy as well as capillary electrophoresis with laser-induced fluorescence detection is employed for the analysis of DNA recovered from solid-phase resins, while the polymerase chain reaction (PCR) is used to evaluate the amplifiable nature of the eluted DNA. We demonstrate that DNA can be directly recovered from white blood cells with an efficiency of roughly 70%, while greater than 80% of the protein is removed with a 500-nl bed volume muSPE process that takes less than 10 min. With a capacity in the range of 10-30 ng/mg of silica resin, we show that the DNA extracted from white blood cells, cultured cancer cells, and even whole blood on the low microliter scale is suitable for direct PCR amplification. The miniaturized format as well as rapid time frame for DNA extraction is compatible with the fast electrophoresis on microfabricated chips.
Tian
H
H
Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.
Hühmer
A F
AF
Landers
J P
JP
D000327:Adsorption; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification*; D004273:DNA, Neoplasm / Q000032:analysis; D006801:Humans; D007962:Leukocytes / Q000737:chemistry; D008904:Miniaturization; D016133:Polymerase Chain Reaction / Q000379:methods; D012117:Resins, Synthetic; D012822:Silicon Dioxide / Q000737:chemistry*; D014407:Tumor Cells, Cultured
D004273:DNA, Neoplasm; D012117:Resins, Synthetic; D012822:Silicon Dioxide; D004247:DNA