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 onto a 3D printed part, which is fixed to the motor. Thanks to this system we can place the magnets close or away from the tubes, allowing us - with the addition of 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 the DNA extraction.
About the magbeads
The coating :
The main goal of the coating is to bind to DNA. The most common coatings are SiO2 and TiO2. In the case of SiO2, the binding with DNA can be controlled through the concentration of salt (ions) in solution: in a solution highly concentrated in salt, the SiO2 coating is positively charged and will therefore attach to DNA thanks to Van der Waals interactions. In a solution with a low concentration in salt, SiO2 will repel DNA because of electrostatic forces.
Magnetic core & Field:
Once the DNA is attached to the beads, we want to move them around. This is done by applying a magnetic field (in our case using permanent magnets). The beads will move from the region with a low magnetic field to the region with a high magnetic field.
About the protocol
Here are 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 solution highly concentrated in salt 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 a first time the medium with an Eluant solution. We remove the Eluant with the electronically controlled pipette (DNA is still located on the edge, bound to the magnetic beads). For this wash, we continue to use a solution with a high salt concentration to keep DNA bound to the beads.
5) The last wash is done using a solution with a low salt concentration and by eating the solution to 70°C: the DNA begins 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, 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.
About the magnetic field
In order to create a magnetic field, we can either use permanent magnets or electromagnets.
Permanent magnets can be really strong, but they need to be physically moved in order to apply or not a magnetic field. On the other hand, electromagnets are weaker but can be turned on and off with a numerical controller.
In order to reduce the amount of troubleshooting, we would prefer electromagnets because no mechanical action is required. However the electromagnets that we tried to use or create were too weak to catch the magnetic beads. Therefore, we had to use permanent magnets.
To move the magnets we use an actuator, which is a motor allowing linear movement. The motor is placed directly on the rotative plate, in order to be sure of the relative position of the magnets compared to the purification tubes. We also added a support system of the piece motor to be sure it slides correctly to the tubes.
To place the magnets as close as possible from the tube we also tailored the lysis aluminum block in this particular way :
REFERENCES
[1] SEPMAG-TOFU-The basic guide for magnetic DNA purification by Alanna Klose https://www.sepmag.eu/blog/bid/387109/3-problems-when-using-classical-magnetic-separation-rack-in-cell-sorting ⇒ also SEPMAG, information on issues one can encounter using magbeads
[2] Magnetic Beads gDNA Kit (Bacteria) Geneaid (extraction protocol using magbeads)