Team:Grenoble-Alpes/purification module
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PURIFICATION MODULE
In our automated system, a crucial step is to extract DNA in order to get through our detection process. Indeed at the end of the lysis step, our solution contains a lot of cellular debris that could prevent the process from being carried out correctly.
A lot of different methods can be used to purify DNA in a lab. The most common example is the use of a silica minicolumn DNA purification.
Looking at all those purifications methods from an engineering point of view, the main common issue is the need for a centrifugation step at some point in the process. As it is difficult to obtain the necessary force to extract DNA in a small low-cost automated system, we looked for an alternative way and that’s how we discovered about magnetic beads.
Magnetic beads are very small (1 µm) particles, consisting of a ferromagnetic core and a coating.
The coating of the beads is used to attach the DNA in the solution, whereas the ferromagnetic core is used to move the beads to the edge of the tube. It allows us to extract DNA by applying a magnetic field following by a few washing steps.
In order to apply the magnetic field, we used small permanent magnets and an actuator. The magnets are crimped in a 3D printed part, which is fixed to the motor. Thanks to this system we can place the magnets closer or further away from the tubes, allowing us with the pipette to automate the DNA extraction.
Click on the button below to get more details about how magnetic beads work, and the different washing steps of a DNA extraction using them.
About the magbeads
The coating :
The main goal of the coating is to bind with DNA. The most commonly used coating are SiO2 and TiO2. In the case of SiO2, the binding with DNA can be control through the concentration of salt (ions) in solution : in a high salt solution the SiO2 coating is positively charged and will therefore attach to DNA thanks to Van der Waals interactions ; in a low salt solution SiO2 will repel DNA because of electrostatic forces.Magnetic core & Field : Once the DNA is attached to the beads, we want to move those around, this is done by applying a magnetic field (in our case through permanent magnet). The beads will move from the region with a low magnetic field to the region with a high magnetic field.
The different steps of magnetic beads DNA purification: 1) Magnetic beads are put into the solution containing a lot of residues and the DNA that we want to extract. We add a high salt solution to enable the binding between the beads and DNA.
2) DNA binds to the beads.
3) a magnetic field is applied to move the beads to the edge.
4) we wash beads removing solution and adding new high salt solution by automatic pipetting. (DNA is still located on the edge through those washing steps) For those washes we continue to use a high salt solution to keep DNA bound to the beads.
5) The final wash is done with a low salt solution : the DNA begin to detach itself from the beads. We remove the magnetic field in order to release all the DNA into the solution.
6) We apply once again the magnetic field (still with a low salt solution), and we pipette the center of the tube : this way we recover the DNA but not the beads. Issues that one can encounter using magnetic beads purification : Sedimentation: beads that are too large will fall to the bottom of the tube, they will no longer be retrievable, this problem is avoided by limiting the time during which the balls are pulled. Pipetting: Good pipetting accuracy is required to avoid inadvertently pipetting beads. A biologist should be careful not to shake when pipetting, a machine should be sufficiently stable and placed well in the center of the hole.