Technology
DNA extraction from the saliva sample is conducted using the automated Thermo Fisher DNA extraction kit.
The purified DNA concentration is meticulously measured to confirm the quality of the extracted DNA.
The purified DNA undergoes amplification using iPLEX extension primers designed for each SNP of interest. All oligos for PCR amplification and iPLEX extension reactions are custom designed by PGx Lab Solutions. Other PCR reagents are exclusively supplied by Agena Biosciences, USA.
Following PCR, excess nucleotides undergo dephosphorylation by shrimp alkaline phosphatase (SAP). This is succeeded by the iPLEX single-base extension reaction, wherein a mix of oligonucleotide extension primers, designed to anneal to the amplified DNA fragments, is added. An extension enzyme and mass-modified dideoxynucleotide terminators are also introduced. The extension primers align directly adjacent to each SNP site for assay, and they are extended and terminated by a single complementary base into the genotyping target site.
The extension products (analytes) undergo desalting using Clean Resin. Subsequently, they are transferred from the microtiter plate via an automated nanodispenser onto a SpectroCHIP® Array. Here, they crystallize with a pre-spotted MALDI matrix.
The SpectroCHIP Array is loaded into the MassARRAY Analyzer. The analyte crystals are irradiated by a laser, inducing desorption and ionization. The positively charged molecules then 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. The results are automatically displayed using Typer software. The entire process, from laser firing to signal detection, takes less than 50 minutes to analyze 384 samples.
