This cost-effective method can find wide applications in molecular diagnosis and screening of FXS and Fragile X-related disorders. It's fast in turnaround time and less investment in equipment. Our PCR-based assay can facilitate classification of full spectrum of the FXS and Fragile X-associated disorders, including intermediate, premutation, and full mutation with robustness and rapid reporting time.
The procedure will be demonstrated by Weng Chua from PerkinElmer Singapore. Start by removing the PCR buffer mix, sample diluent, and DNA samples from the 20 degree Celsius freezer, and leaving them at room temperature for 20 to 30 minutes to thaw. Prior to using the reagents, vortex and briefly spin them down.
Measure the DNA sample concentration with a spectrophotometer, and if required, dilute it to 25 nanograms per microliter with sample diluent. Label the wells of a PCR plate to identify reference and tested DNA samples and calculate the number of reactions needed for the test, reference, and negative control samples. Prepare the PCR master mix by combining 15 microliters of PCR buffer mix to 0.6 microliters of sample diluent and 0.4 microliters of polymerase for each reaction.
Vortex the mix and spin it down. Then dispense 18 microliters of the mixture into each well. Then pipette two microliters of each DNA into the appropriate well.
Mix the reagents by pipetting up and down five times, then seal the plate. Place the plate in the thermocycler and run the PCR according to the manuscript directions. Preheat the incubator shaker to 65 degrees Celsius and add 80 microliters of 1X TE buffer to each PCR product.
Use a multichannel pipette to transfer the samples to a PCR cleanup plate and put the plate in the shaker. Incubate it at 65 degrees Celsius while shaking at 1200 RPM for 10 minutes. After the incubation, cool the incubator shaker down to 25 degrees Celsius, set the vacuum instrument to 250 millibar, and aspirate the solution through the filter.
After 15 minutes, the wells should have no liquid remaining. Turn off the vacuum and add 50 microliters of 1X TE buffer to each well. Aspirate the solution for 10 minutes using the previous vacuum settings.
Dry the bottom of the filter plate by pressing it firmly on a stack of paper towels and then add 20 microliters of 1X TE buffer to the bottom center of each well. Place the plate in the shaker and incubate at 25 degrees Celsius while shaking at 1200 RPM for five minutes. After the incubation, transfer at least 15 microliters of the purified PCR product to a fresh 96 well PCR plate.
Prior to starting, bring the DNA dye concentrate, DNA gel matrix, DNA marker, DNA ladder, and purified DNA samples to room temperature. Set up the priming station by replacing the syringe and adjusting the base plate, then release the lever of the syringe clip and slide it to the top position. Start the sizing software and prepare the gel dye mix.
Vortex the dye concentrate for 10 seconds, then spin it down. Then, add 25 microliters of the dye to a gel matrix vial and vortex the solution to mix. Transfer the gel dye mix to a spin filter, place it in the centrifuge, and spin it for 10 minutes at 1500 times g.
When ready to load the gel dye mix, insert a new DNA chip into the priming station and add nine microliters of the gel dye mix into the well that is marked with G.Close the priming station and ensure that the plunger is positioned at the one milliliter mark. Press the syringe plunger down until it is held by the clip. Wait for exactly 30 seconds, and then release the clip.
Wait for five more seconds, and then slowly pull the plunger back to the one milliliter position. Open the priming station and add nine microliters of gel dye mix into the wells marked with G.Add five microliters of marker into the well marked with a ladder symbol and each of the 12 sample wells. Add one microliter of the ladder to the well with the ladder symbol and one microliter of the PCR product or water to the sample wells.
Vortex the chip for one minute at 2400 RPM. Insert it into the bioanalyzer and run the chip within five minutes. When the run is complete, export the peak data as csv table files.
A premutation female sample and a full mutation female sample are used as reference samples. An upper and lower marker peak are included in the fragment size profile, and there is usually a primer complex peak at about 95 base pairs. These reference samples are used to construct a regression standard curve.
The linear regression standard curve is then used to calculate repeat sizes of unknown samples. Clinical normal, intermediate, premutation, full mutation, and mosaic full mutation samples can be correctly classified with this method. In some cases, only one peak is displayed in the microfluidic electrophoresis results, which is likely due to the presence of normal homozygous alleles.
One type of suboptimal result is baseline bias, which can be ambiguous or uninterpretable, and is suspected to be caused by instrument malfunction. When attempting this procedure, it is important to ensure appropriate amount of DNA and quality before starting the protocol. FMR1 gene sequencing can be performed to identify the AGG interruption pattern.
And methylation-sensitive restriction enzyme or bisulfide modification assay can be used to monitor the methylation status. This is a fast, robust, and cost-effective technique. It will assist other researchers in conducting a population-based study on the carrier status of FXS and Fragile X-associated disorders.
