Wild-type Blocking PCR Combined with Sanger Sequencing for Detection of Low-frequency Somatic Mutation

This article introduces the application of a low-frequency detection method based on Sanger sequencing in angioimmunoblastic lymphoma. Provide a basis for applying this method to other diseases.

We are interested in understanding low-frequency somatic mutation in cancer tissues. This article introduces the application of a low-frequency mutation detection method based on our Sanger sequencing in angioimmunoblastic lymphoma test, providing a basis for applying this method to other disease tests. Currently, there are low-frequency mutation detection methods such as quantitative PCR, digital PCR, and NGS.

But there's no report on detection of IGHV7-3 low frequency mutation based on Sanger sequencing, of which detection limit is only 5%to 20%Generally due to technical limitations, low-frequency detection based on Sanger sequencing cannot be accurately quantified. Our technique offers high sensitivity, low cost, and strong flexibility, making it an ideal choice for low-frequency mutation detection. We are planning to design pseudogene primers as well as multiplex GCR primers to explore their application in blood diseases.

To begin, prepare all the reagents required for tissue sample preparation and DNA extraction. Add 500 microliters of Deparaffin buffer and 20 microliters of proteinase K solution to about 30 milligrams of tissue in a microcentrifuge tube. Vortex the microcentrifuge tube for at least 10 seconds and place it in a dry thermostat set at 55 degrees Celsius for 90 minutes.

After incubation, transfer the sample including liquid and tissue to the spin column that is placed in a filter tube. Centrifuge at 16, 500 g for five minutes to separate the liquid into the filter tube. Then, transfer the liquid from the filter tube to a labeled sample tube and perform DNA extraction according to the manufacturer's instructions.

Measure the concentration and purity of the extracted DNA. To begin, extract the DNA from Formalin-Fixed Paraffin-Embedded tissue samples. Then, centrifuge the primer dry powder at 5, 400 to 8, 400 g for two to three minutes, and add the appropriate volume of double distilled water along the wall of the primer tube.

Add five microliters each of the forward and the reverse primer to 50 microliters of wildtype primer stock, and 40 microliters of double distilled water. After mixing two to three times, vortex the tube and centrifuge it briefly. For the polymerase chain reaction or PCR procedure, add the required reagents to each reaction tube.

Vortex to mix and centrifuge briefly. Place the PCR tube in the PCR instrument for amplification. Set the thermal cycle program and run it.

Then, perform gel electrophoresis on the PCR product using 2%agarose to check whether the target fragment is amplified. To purify the PCR products, briefly centrifuge the purificase I and purificase II that have been brought to room temperature and prepare the purification reaction mixture. Place the PCR tube in the PCR instrument and run the machine with the preset parameters.

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 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.