Genetics
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Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry
Chapters
Summary June 21st, 2018
Identification of genetic variants contributing to complex human disease allows us to identify novel mechanisms. Here, we demonstrate a multiplex genotyping approach to candidate genes or gene pathway analysis that maximizes the coverage at low cost and is amenable to cohort-based studies.
Transcript
This method can help answer key questions in the clinical diagnostics and genetics fields, such as detecting genetic markers associated with disease outcomes, included the human papilloma virus. The main advantage to this technique is that it's a cost effective targeted and time efficient diagnostic method. To begin this procedure, reconstitute any ordered lyophilized primers with molecular-grade water.
Pull the forward and reverse primers for each multiplexed assay into a stock primer mix, such that it contains each primer at a concentration of zero-point-five millimolar. After this, prepare a master mix for the PCR as outlined in the text protocol. Add four microliters of this mix to each well of a 384-well PCR plate, along with one microliter of genomic DNA at a concentration between five and 10 nanograms per microliter.
Cover the plate with a plate cover, and centrifuge for 200 times G for one minute at room temperature. Then run the PCR as outlined in the text protocol. Load one microliter of PCR product with three microliters of loading buffer on a 2%agarose gel.
Run at 100 volts for 45 minutes to ensure that the DNA amplification was successful. Next, prepare an SAP master mix as outlined in the text protocol. Add two microliters of the freshly made SAP master mix to each well of the previously run PCR plate.
Centrifuge briefly at 1, 000 times G for one minute. And then return the plate to the thermocycler. Run the thermocycler at 37-degrees Celsius for 40 minutes.
And then at 85-degrees Celsius for five minutes to inactivate the enzyme. After calculating the dilution factor for each primer, combine the determined volumes of each to make up a primer pool. Add water as necessary to dilute this pool.
Assemble the extension master mix for either the high plex assay or the low plex assay, as outlined in the text protocol. Add two microliters of the extension master mix to each well of the plate, bringing the total volume in each well up to nine microliters. Centrifuge briefly at 1, 000 times G for one minute, and then return the plate to the thermocycler.
After this, add 16 microliters of deionized water to each well, bringing the total volume up to 25 microliters per well. Apply six milligrams of resin evenly across an entire dimple resin plate. Let the resin dry for 20 minutes at room temperature.
Then, apply the resin to the reaction plate. Using a suspension mixer, rotate the plate at a slow speed for five minutes at room temperature. Next, centrifuge the plate at 3, 200 times G for five minutes to avoid applying the resin to the mass spectra chip.
Upload the plate's sample layout to the system's software interface. Using a dispensing system, apply the genotyping analyte mixture from the plate to the genotyping chip. Load the chip into the MALDI-TOF system to generate the mass spectra from the analyte mixture, and interpret it using the platform's software interface.
In this study, multiplex genotyping is accomplished using mass spectrometry. Representative SNP data is generated and analyzed for the candidate gene IL13 in a discovery clinical cohort phenotyped for food allergy. One variant, rs1295686, is associated with challenge-proven food allergy.
This is association is confirmed in a replication cohort using the multiplex genotyping approach demonstrated in this protocol. A meta-analysis of the results from the two cohorts provides strong evidence of the association between IL13 with FA.While attempting this procedure, it's important not to contaminate any samples or reagents. It's also important to prepare an extra 10%master mix for each PCR in order to account for any pipetting error.
Following this procedure, other methods like real-time PCR can be performed in order to answer additional questions such as whether gene expression correlates with the identified disease-associated genetic markers. After it's development, this technique paved the way for researchers in the fields of clinical diagnostics and genetics to explore disease associations in patient cohorts. Don't forget that when working with genotyping reagents and the MALDI-TOF system can be hazardous, and precautions such as wearing gloves and a lab coat should always be taken when performing this procedure.
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