DNA separation by silica adsorption
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DNA Separation by Silica Adsorption is an important method of DNA separation that is used in novel technologies that use micro-channels. The principle behind this type of separation relies on DNA molecules binding to silica surfaces in the presence of certain salts and under certain pH conditions.
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[edit] Why is this method of purification important?
Conventional methods for DNA extraction, such as centrifugation with ethanol or preparations made from a commercial purification products like Qiagen columns, 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. Thus conventional methodologies do not meet the rising demands of new technologies. DNA extraction on microchips provides a fast, cost effective, and effective for High-throughput screening, which also has a very small footprint. Developments within this type of extraction have useful applications for biosensors, “lab on a chip” devices, and other new technologies that require rapid, high quality DNA extraction in the field at minimal cost.
[edit] How does this method of purification work?
Before going into great detail, let’s first outline the basic steps of this purification method:
1. Sample is run through micro-channel
2. DNA binds to the channel, all other molecule remain in buffer solution
3. Channel is washed of impurities
4. An elution buffer removes DNA from channel walls and DNA is collected at the end of the channel.
A sample (this may be anything from purified cells to a tissue specimen collected in the field only moments earlier) 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 are shown to occur in the presence of buffer solution with pH at or below pKa of the surface silanol groups.
Although the exact method for this interaction is not well known, 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 better explanation of how DNA binds to silica is based on the action of Guanidium HCl (GuHCl), which acts is a chaotrope. A chaotrope denatures biomolecules by disrupting the shell of hydration around them. This allow a positively charged ion 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 EtOH, 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. Once these are washed from the DNA, the DNA is washed to remove any excess waste particles from the sample and then eluted from the channel using an elution buffer for further Downstream processing. See the figure to the right to understand the DNA profile during the loading, washing, and eluting steps.
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
[edit] What kind of silicon micro DNA extraction surfaces exist?
Methods using silica beads and silica resins have already been created which successfully isolated DNA molecules which can then be PCR amplified. However, these methods have a few problems associated with them. 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 (since beads or resins don’t need to be introduced post manufacturing the chip). One of the possible explanation of DNA-silica interacting mechanism is described here: http://www.bio.davidson.edu/courses/Molbio/MolStudents/spring99/lauren/geneclean.html
[edit] References
- 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 Matricies 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).
