DNA separation by silica adsorption
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DNA separation by silica adsorption is an method of DNA separation that is based on DNA molecules binding to silica surfaces in the presence of certain salts and under certain pH conditions.
Conventional methods for DNA extraction, such as centrifugation with ethanol or preparations using commercial purification kits, cannot be integrated onto microchips because they require multiple hands-on processing steps. In addition, they also require large equipment and high volumes of reagents and samples. DNA extraction on microchips provides a fast, cost effective, and effective for high-throughput screening, which also has a very small footprint. This new method has useful applications for biosensors, “lab on a chip” devices, and other new technologies that require rapid, high quality DNA at minimal cost.
There are two basic steps:
- The sample is run through a micro-channel
- DNA binds to the channel, and all other molecules remain in the buffer solution
- The channel is washed free of impurities
- An elution buffer removes the DNA from channel walls, and the DNA is collected at the end of the channel.
In the actual operations, a sample (this may be anything from purified cells to a tissue specimen) is placed into the chip and lysed. The resultant mix of proteins, DNA, phospholipids, etc., is then run through the channel where the DNA is adsorbed by silica surface in the presence of solutions with high ionic strength. The highest DNA adsorption efficiencies occur in the presence of buffer solution with a pH at or below the pKa of the surface silanol groups. Although the mechanism is not fully understood, one possible explanation involves reduction of the silica’s surface’s negative charge due to the high ionic strength of the buffer. This decrease in surface charge leads to a decrease in the electrostatic repulsion between the negatively charged DNA and the negatively charged silica. Meanwhile, the buffer also reduces the activity of water by formatting hydrated ions. This leads to the silica surface and DNA becoming dehydrated. These conditions lead to an energetically favorable situation for DNA to adsorb to the silica surface.
A further explanation of how DNA binds to silica is based on the action of guanidium HCl (GuHCl), which acts as a chaotrope. A chaotrope denatures biomolecules by disrupting the shell of hydration around them. This allows positively charged ions to form a salt bridge between the negatively charged silica and the negatively charged DNA backbone in high salt concentration. The DNA can then be washed with high salt and ethanol, and ultimately eluted with low salt. After the DNA is adsorbed to the silica surface, all other molecules pass through the column. Most likely, theses molecules are sent to a waste section on the chip, which can then be closed off using a gated channel or a pressure- or voltage-controlled chamber. The DNA is then washed to remove any excess waste particles from the sample and then eluted from the channel using an elution buffer for further downstream processing.
The following solutions have been proposed and validated for use in this process:
DNA binding: GuHCl- based loading buffer
Channel Wash: 80% isopropanol
DNA elution: TE at pH 8.4
Silicon micro DNA extraction surfaces
Methods using silica beads and silica resins have been created which successfully isolated DNA molecules which can then be PCR amplified. However, these methods have associated problems: First, beads and resins are highly variable depending on how well they are packed and are thus hard to reproduce. Each loading of a micro-channel can result in a different amount of packing and thus change the amount of DNA that adsorbed to the channel. Furthermore, these methods result in a two step manufacturing process.
Silica structures are a much more effective method of packing material because they are etched into the channel during its fabrication and is thus the result of a one step manufacturing processes via soft lithography. Silica structures are therefore easier to use in highly parallelized designs than beads or resins.
- Cady, et al. Nucleic acid purification using microfabricated silicon structures. Biosensors and Bioelectronics 19, 59-66 (2003).
- K. A. Melzak, C. S. Sherwood, R. F. B. Turner, C. A. Haynes. Driving Forces for DNA Adsorption to Silica in Perchlorate Solutions. J Colloid and Interface Science 181, 635–644 (1996).
- Tian, et al. Evaluation of Silica Resins for Direct and Efficient Extration of DNA from Complex Biological Matrices in a Miniaturized Format.Analytical Biochemistry 283, 175-191 (2000).
- Wolfe, et al. Toward a microchip-based solid-phase extraction method for isolation of nucleic acids. Electrophoresis 23, 727-733 (2002).
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