This method can help answer key questions in the genetic testing field, such as detection of mosaicism, and liquid biopsy-based genetic testing. The main advantage of this technique is high-throughput detection of low-variant allele fraction. The implications of this technique extend towards diagnosis of tumors, because detecting oncogenic mutations with high sensitivity can contribute to precision medicine.
Start this experiment by adding 10 L of DNA sample buffer to the tubes of an 8-tube strip. Then add 1 microliter of DNA ladder to the first tube of the 8-tube strip, and one microliter of sample genomic DNA to each of the remaining tubes. Use microplate attachment to mix contents of the 8-tube strip by vortexing for one minute.
Place the strip, pipet tips, and a gel device into an electrophoresis instrument. Press start button to start the run. After the complete run, check the electropherogram on the display to confirm that the lower marker contained in the DNA sample buffer is correctly assigned on the electropherogram.
If not, manually assign the marker in the electropherogram mode of the software. Then, in the region mode of the software, specify the size region of the genomic DNA to automatically calculate the concentration of DNA. Add previously-designed primers, probes, and genomic DNA to a new 8-tube strip to achieve a total volume of 15 L.Pipette up and down to mix.
Add the loading platform onto the chips built in the fresh 8-tube strip, and then place the tube strip in the autoloader. Make sure that there is a contact between the chips and the loading platform. Next, place a loading slider in the platform, and use a stopper to hold the slider off of the loader.
Pipette 15 L of the PCR mixture near the tip of the slider, and then press the loader button to run the loader for one minute. Remove the tube strip from the loader after the run, and place it in sealing enhancer. Carefully push the slide lid and the edge of the top lid.
Run the sealing enhancer for approximately two minutes. If sealing is incomplete, indicated by a puddle of liquid, repeat the run for an additional minute. Add 230 L of sealing fluid to the tubes.
Place the tube strip in the thermal cycler, and run the PCR as described in the protocol. If there is an uneven distribution of positive partitions, adjust the temperature or the duration of the PCR. To detect and analyze fluorescence intensity of the PCR products, place the tube strip on the detection jig, and add 6 mL of distilled water.
Remove any visible air bubbles using a pipette tip. Load the jig into the detector. In the detection software, select Fluorescence, Experiment, and then Sample NTC tabs, and click the RUN button to start the run.
After the complete run, confirm the position plot, histogram, and 2-D scatter plot. To collect the PCR product, remove the sealing fluid from the tube. Add 100 L of TE buffer, and vortex vigorously for 30 seconds.
Briefly centrifuge the tubes in a tabletop centrifuge, and proceed as described in the text protocol to finish collecting the PCR product. Position plots created by digital PCR show yellow HEX fluorescence in both patients and father samples, indicating presence of APC allele variant T, but not in no template control. Only patients genomic DNA contains C-variant, as shown by a green fluorescence of FAM.
As confirmed further by scatter plots, both patient and father are positive for HEX, or variant T, whereas only the patient shows the presence of variant C.Patients variant allele fraction, or VAF, calculated by DPCR, was 13.2%similar to 12.7%achieved by next generation sequencing. On the other hand, the VAFs of the patients'parents and healthy donors were less than 0.1%Electropheresis of collected and concentrated DPCR products confirm the size of the predicted fragment to be 123 base pairs in patients'and parents'DNA. While attempting this procedure, it is important to remember to keep the bench clean by, for example, using a fan filter unit.
