To begin, perform polymerase chain reaction, or PCR, to amplify the desired region of the tissue-extracted DNA. Bring the sequencing amplification enzyme master mix, buffer, and double-distilled water to room temperature and spin off instantly. After preparing the tubes for sequencing, place the PCR tube on the preset PCR instrument for amplification.
Then remove the product from the PCR instrument and store the reaction products in the refrigerator, protected from light. Vortex the magnetic beads thoroughly. Add 10 microliters of magnetic beads and 40 microliters of 80%ethanol to the 96-well plate and seal the plate with a film.
Place the 96-well plate on a vortex shaker for 10 seconds to fully suspend and mix the beads. Then secure the plate with a rubber band on a horizontal oscillator and vibrate at the maximum setting for three to five minutes. Next, place the PCR plate on a magnetic stand, ensuring it is firmly pressed.
Keep the stand at four degrees Celsius for three minutes for static adsorption. Once the beads are adsorbed, remove the ceiling film and pour out the waste liquid. Rinse the beads twice with 40 microliters of 80%ethanol.
After pouring off the waste liquid, place the plate on absorbent paper and gently pat it dry. Place the absorbent paper with the magnetic plate into a centrifuge and spin at 120 g briefly. After removing the alcohol, place the plate in an oven at 60 degrees Celsius for three to five minutes to dry the beads completely.
Next, add 40 microliters of pure water to the plate and seal it with a film. Shake it on a vortex shaker for three to five seconds and place the plate on a horizontal shaker. Secure it and let the plate shake for three to five minutes to dissolve the purified sequencing product.
Finally, spin the dissolved sequencing product at 180 g and use the product for genomic analysis using capillary electrophoresis. This method could detect the known RHOA G17V mutation and other low-frequency mutations in the amplification interval of upstream and downstream primers. Mutations in two additional genes, namely, IDH2 and JAK1, were also correctly analyzed using this method.
