Personalized Prescribing Inc. processes the Rx-Report™ test samples at their own laboratory in the Greater Toronto Area. Once the test samples (saliva) reach the laboratory, the process starts.
DNA is extracted from the saliva sample using an automated Thermo Fisher DNA extraction kit.
The purified DNA concentration is measured to confirm the quality of the DNA extracted.
The purified DNA is amplified by using iPLEX extension primers for each SNP or somatic mutation of interest. All oligos for PCR amplification and iPLEX extension reactions are custom designed by Agena Bioscience.
After the PCR amplification, excess nucleotides are dephosphorylated by shrimp alkaline phosphatase (SAP). This is followed by the iPLEX single base extension reaction in which a mix of oligonucleotide extension primers, designed to anneal to the amplified DNA fragments, is added together with an extension enzyme and mass-modified dideoxynucleotide terminators. The extension primers anneal directly adjacent to each SNP site to be assayed and are extended and terminated by a single complementary base into the genotyping target site.
The extension products (analytes) are desalted using Clean Resin and then transferred from the microtiter plate via manual dispensation or an automated nanodispenser onto a SpectroCHIP® Array, where they crystalize with a pre-spotted MALDI matrix.
The SpectroCHIP Array is loaded into the MassARRAY Analyzer, where the analyte crystals are irradiated by a laser, inducing desorption and ionization. The positively charged molecules accelerate into a flight tube towards a detector. Separation occurs by time-of-flight which is proportional to the mass of the individual molecules. After each laser pulse, the detector records the relative time of flight for each extension product and the results are displayed automatically using Typer software. The entire process from laser firing to signal detection takes less than 50 minutes to analyze 384 samples.