This article describes a method for purifying nucleic acids from human blood using magnetic glass particles (MGPs). The process involves lysing cells, binding nucleic acids to MGPs, and eluting the purified nucleic acids for downstream applications.
Take a tube containing freshly drawn human blood and add a lysis buffer.
The detergent in the buffer permeabilizes the cell membrane, lysing the cells and releasing intracellular nucleic acids, or NAs, and nucleases.
Chaotropic agents in the buffer denature nucleases, preventing NA degradation.
Add magnetic glass particles or MGPs — silica-coated magnetic particles — and mix thoroughly.
The buffer's low pH protonates silica, rendering a positive charge on the MGPs' surface.
Negatively charged NAs bind electrostatically to the charged surface, forming an NA-MGP complex.
Under a magnetic field, NA-MGP complexes collect at the side of the tube.
Discard the supernatant containing cell debris and proteins.
Perform multiple washing steps by resuspending the NA-MGP complexes in a wash buffer, applying a magnetic field to collect the complexes, and discarding the supernatant with the remaining contaminants.
Resuspend complexes in an elution buffer. The high pH disrupts NA-MGP interaction, releasing the NAs. The eluted NAs are ready for downstream assays.
To purify nucleic acids from the collected blood, again mix the contents of the tube by flipping the vacuum tube by hand 5 to 10 times. Pour the prepared aliquot of MGPs directly into the blood collection tube, then, place a new lid from an unused blood collection tube on the tube containing the sample. Place a finger on the lid to ensure that the tube is tightly closed, and mix the contents of the tube by flipping 5 to 10 times.
Place the tube in the magnetic holder and keep a finger on the lid to ensure the tube is tightly closed. Then, flip the magnetic holder with the tube a few times to disperse the MGPs before they collect on the side of the tube with the magnet. Remove the lid of the tube and discard the contents of the tube into a 50-milliliter collection tube using a disposable pipette.
Pour the prepared aliquot of WB1 directly into the blood collection tube, and close the lid on the tube. Mix the content by hand 5 to 10 times. Then, collect the beads at the side of the tube using the magnet, and remove the liquid as before. Resuspend the beads in the prepared aliquot of WB2. Then, discard the solution, and repeat the procedure with the prepared aliquot of WB3.
Finally, remove the tube from the magnetic holder and pour the prepared aliquot of EB directly into the blood collection tube. Place the lid on the tube, and resuspend the MGPs by tapping 5 to 10 times with a finger. Finally, use a 1.5-milliliter disposable pipette to transfer one droplet of the resuspended MGPs to the downstream nucleic acid amplification reaction mix.
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