Our research team is devolving into the elements that dictate the sensitivity and resistance to 5-fluorouracil, a key chemotherapeutic agent for gastric cancer, emphasizing genetic inferences. We are dedicated to uncovering how genetic differences impact drug reactions and assessing the feasibility of tailoring treatment to individual genetic profiles. This protocol is tailored to detect single-nucleotide polymorphisms in patients with gastric cancer, elucidating the correlation between genetic mutations and their sensitivity to fluorouracil chemotherapy.
This breakthrough enables the prediction of patients'responses to fluorouracil, facilitating the customization of treatment strategies. This protocol stands out by offering high sensitivity for various sample types, including blood, reducing invasiveness with multiple samples. Utilizing semiconductor sequencing simplifies operations and consequently time by directly detecting ion changes by passing fluorouracils.
To begin, take the nucleic acids extracted from the paraffin embedded gastric cancer tissue samples. Place components of the custom made gastric cancer multi-gene joint detection kit on ice. After mixing the components thoroughly, centrifuge them for 10 seconds, then add reagents into a 0.2 milliliter PCR tube sequentially, and mix for five seconds by vortexing.
Place each reaction tube on the thermal cycler and run the amplification program for DNA samples and cDNA products. Thaw the primer digestion enzyme on ice. Add two microliters of primer digestion enzyme to each reaction tube.
After vortexing and centrifuging the tubes, place them on the thermal cycler and run the digestion program. Thaw the adapter connecting reagents on ice. Label a 1.5 milliliter microcentrifuge tube as adapter mixture X and add the required components.
Take the digested primer product from the thermal cycler. Add the ligation reagents to the tube, vortex for five seconds, and centrifuge at low speed for 10 seconds at 2, 500 G.Then place the tube on the thermal cycler and run the amplification program for DNA samples and for cDNA products. To begin, remove the DNA purification magnetic beads from the two to eight degrees Celsius refrigerator.
Centrifuge the beads for 10 seconds at 2, 500 G after mixing. Transfer the adapter ligated reaction product to a 1.5 milliliter low absorption microcentrifuge tube. Add 45 microliters of DNA purified magnetic beads to each tube.
After vortexing the components, centrifuge the tube for 10 seconds. Then place the tube on a magnetic rack for three minutes. Carefully discard the supernatant without pipetting the beads.
Now transfer 300 microliters of 75%freshly prepared ethanol into each microcentrifuge tube. Gently rotate the tubes four times at 180 degrees. Once the solution clears, promptly discard the supernatant while avoiding pipetting out the beads.
Then, centrifuge the tubes briefly after removing the tubes from the magnet rack. Place the tubes on the magnet rack and pipette any remaining liquid. Open the caps of the 1.5 milliliter microcentrifuge tubes and allow the beads to dry at room temperature for five minutes.
After thawing and vortexing the PCR related reagents, centrifuge the reagents for 10 seconds. Remove the 1.5 milliliter microcentrifuge tube from the magnet rack and add PCR reagents. Centrifuge the vortex tube briefly to avoid liquid drops on the tube wall and cover.
Then transfer the PCR product into a new PCR tube. Incubate on a thermal cycler, and run the amplification program for DNA and cDNA samples after incubation centrifuge to the PCR tube for 10 seconds and transfer the product into a new 1.5 milliliter low absorption microcentrifuge tube. Add 25 microliters of DNA purified magnetic beads to each tube and mix well by vortexing.
Centrifuge the tube at low speed and incubate at room temperature for five minutes. Then place the tubes on a magnetic rack for three minutes until the solution clears. Transfer the supernatant to new microcentrifuge tubes, avoiding pipetting out the beads.
Transfer 300 microliters of 75%freshly prepared ethanol into the tubes and wash the beads two times using a magnetic rack as demonstrated earlier. After centrifuging the tubes briefly, place the tubes on the rack and pipette out any remaining liquid. Open the caps of 1.5 milliliter tubes and dry the beads at room temperature for five minutes.
Pipette 50 microliters of eluent into the tubes. Perform a brief centrifugation after vortexing the tubes. Place the tubes on a magnetic rack for three minutes until the solution becomes clear.
Carefully transfer the liquid into a new tube and label it with the library name.
