DNA separation by silica adsorption
Encyclopedia
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.
, 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.
1. The sample is run through a micro-channel
2. DNA binds to the channel, and all other molecules remain in the buffer solution
3. The channel is washed of impurities
4. An elution buffer removes the DNA from channel walls, and the DNA is collected at the end of the channel.
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 are shown to occur in the presence of buffer solution with a pH at or below the pKa of the surface silanol groups.
Although the exact mechanism for this interaction is not well 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 better 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
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
Why is this method of purification important?
Conventional methods for DNA extractionDNA extraction
DNA isolation is a routine procedure to collect DNA for subsequent molecular or forensic analysis. There are three basic and two optional steps in a DNA extraction:...
, such as centrifugation
Centrifugation
Centrifugation is a process that involves the use of the centrifugal force for the sedimentation of mixtures with a centrifuge, used in industry and in laboratory settings. More-dense components of the mixture migrate away from the axis of the centrifuge, while less-dense components of the mixture...
with ethanol
Ethanol
Ethanol, also called ethyl alcohol, pure alcohol, grain alcohol, or drinking alcohol, is a volatile, flammable, colorless liquid. It is a psychoactive drug and one of the oldest recreational drugs. Best known as the type of alcohol found in alcoholic beverages, it is also used in thermometers, as a...
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
High-throughput screening
High-throughput screening is a method for scientific experimentation especially used in drug discovery and relevant to the fields of biology and chemistry. Using robotics, data processing and control software, liquid handling devices, and sensitive detectors, High-Throughput Screening allows a...
, 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.
How does this method of purification work?
Before going into great detail, let’s first outline the basic steps of this purification method:1. The sample is run through a micro-channel
2. DNA binds to the channel, and all other molecules remain in the buffer solution
3. The channel is washed of impurities
4. An elution buffer removes the DNA from channel walls, and the DNA is collected at the end of the channel.
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
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
, 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 a pH at or below the pKa of the surface silanol groups.
Although the exact mechanism for this interaction is not well 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 better 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
Downstream processing
Downstream processing refers to the recovery and purification of biosynthetic products, particularly pharmaceuticals, from natural sources such as animal or plant tissue or fermentation broth, including the recycling of salvageable components and the proper treatment and disposal of waste. It is an...
.
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
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
Soft lithography
200px|right|thumb|Figure 1 - "Inking" a stamp. PDMS stamp with pattern is placed in Ethanol and ODT solution200px|right|thumb|Figure 2 - ODT from the solution settles down onto the PDMS stamp. Stamp now has ODT attached to it which acts as the ink....
. 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
See also
- BiosensorBiosensorA biosensor is an analytical device for the detection of an analyte that combines a biological component with a physicochemical detector component.It consists of 3 parts:* the sensitive biological element A biosensor is an analytical device for the detection of an analyte that combines a biological...
- Lab-on-a-chipLab-on-a-chipA lab-on-a-chip is a device that integrates one or several laboratory functions on a single chip of only millimeters to a few square centimeters in size. LOCs deal with the handling of extremely small fluid volumes down to less than pico liters. Lab-on-a-chip devices are a subset of MEMS devices...
- Microelectromechanical systemsMicroelectromechanical systemsMicroelectromechanical systems is the technology of very small mechanical devices driven by electricity; it merges at the nano-scale into nanoelectromechanical systems and nanotechnology...
- MicrofluidicsMicrofluidicsMicrofluidics deals with the behavior, precise control and manipulation of fluids that are geometrically constrained to a small, typically sub-millimeter, scale.Typically, micro means one of the following features:* small volumes...
- BiochipBiochipThe development of biochips is a major thrust of the rapidly growing biotechnology industry, which encompasses a very diverse range ofresearch efforts including genomics, proteomics, and pharmaceuticals, among other activities...
- Downstream processingDownstream processingDownstream processing refers to the recovery and purification of biosynthetic products, particularly pharmaceuticals, from natural sources such as animal or plant tissue or fermentation broth, including the recycling of salvageable components and the proper treatment and disposal of waste. It is an...