This budget-friendly magnetic bead-based DNA extraction protocol makes high throughput sequencing accessible to resource limited labs and studies. This protocol does not require expensive equipment for high quality DNA extraction and it can be helpful in certain diagnostic or research situations where obtaining a sufficient quantity of DNA with a quality for high throughput sequencing is challenging. This protocol is easy to replicate with a one-time demonstration.
It should be tried with a small number of samples first to get familiar with the workflow. To begin, hydrate the mosquito tissue samples stored in greater than 70%alcohol by adding 100 microliters of PCR grade water and incubating for one hour at four degrees Celsius to soften the tissue. After the incubation, discard the water and add 100 microliters of Proteinase K buffer enzyme mix.
Then use a microcentrifuge tube pestle to homogenize the tissue. After homogenization, centrifuge the tissue lysate and incubate for two to three hours at 56 degrees Celsius. To extract DNA, pipette 100 microliters of the tissue lysate into a new clean microcentrifuge tube.
Then add 215 microliters of the magnetic bead master mix. Using a pipette, mix the lysate and the magnetic beads mixture for 10 to 20 seconds, then let it stand for 10 minutes at room temperature. During the incubation, gently shake the tube occasionally to maximize the binding of the DNA to the magnetic beads.
Next, place the tube on the magnetic bead separator until the solution becomes clear. Then using a pipette, gently aspirate the liquid from the tube without disturbing the magnetic beads holding the DNA. Move the tube away from the magnetic bead separator and wash the beads by adding 325 microliters of washing buffer one.
Mix thoroughly by pipetting and incubate for one minute at room temperature. After the incubation, place the tube on the magnetic bead separator and remove the supernatant as previously demonstrated, then move the tube away from the magnetic bead separator and wash the beads once with washing buffer one. After the second wash with buffer one, wash the beads twice with 250 microliters of washing buffer two in the same manner.
After the last wash, move the tube away from the magnetic bead separator and add 100 microliters of elution buffer. Mix thoroughly by pipetting and then incubate the tube for two minutes at room temperature before placing it back on the magnetic separator. When the solution becomes clear, transfer the supernatant into a new, clean 0.5 milliliter microcentrifuge tube and store appropriately.
Shown here as a typical microvolume fluorometer reading of the mosquito DNA in an elution buffer containing 0.5 millimolar EDTA. The average 260 to 280 nanometer absorbance ratio is 2.3. This table shows the costs and the number of samples processed in one working day for different extraction methods.
The typical reagent and consumable cost for an extraction by this protocol is around 9.50 per sample. This cost is equivalent to any typical magnetic bead-based extraction method. The major cost benefit of this protocol comes from not requiring the automated DNA extraction instrument.
Make sure to change pipette tips between samples to prevent cross-contamination of DNA when working with multiple samples. We use the high quality DNA extracted using this method for whole genome sequencing. We are currently using the sequencing data to identify novel mutations in insecticide resistance or immune genes of concern in public health and disease control.
Having affordable solutions for high throughput sequencing opens exciting doors for population genomics and landscape genomics aimed at understanding mosquito dispersal and gene flow which influences the success of many novel genetic control strategies.
