Oragene dna og 250

Genotyping of NPHSII samples was performed on DNA extracted from blood and carried out using Taqman (Applied Biosystems, Life Technologies, Carlsbad, CA, USA) and KASPar (LGC, Teddington, UK) genotyping assays as well as restriction fragment length polymorphism (RFLP) analysis. For the CoRDia SMI + RR arm, saliva was collected using the Oragene-DNA OG-250 and DNA manually purified using DNA Genotek’s PrepIt-L2P DNA extraction kit (DNA Genotek Inc., Ontario, Canada). Individuals in the CoRDia SMI + RR arm and selected NPHSII samples (n = 185) were genotyped using the Cardiac Risk Prediction Array (Randox Laboratories Ltd, Crumlin, Co Antrim, UK) according to the manufacturer’s instructions. The Cardiac Risk Prediction Array is a multiplex SNP genotyping system which uses Randox’s Biochip Array Technology [13 (link)] to genotype 19 CHD risk SNPs. The protocol involves using multiplex PCR to amplify target DNA in an allele-specific manner. Amplicons are detected by hybridisation to spatially tethered probes on the biochip array surface. Each position on the biochip array corresponds to a specific allele and genotypes are determined using the Evidence Investigator Analyser. In some instances (e.g. for rare genotypes) Sanger sequencing was used to confirm the result.

After providing signed informed consent, pharmacists collected a 2mL saliva sample from participating patients using the Oragene DNA OG-250 (DNA Genotek Inc). The samples were transported to the PGx laboratory in Leiden University Medical Center by research staff or mail. DNA was extracted in accordance to Oragene DNA OG-250 isolation procedure, where a solution volume of 100µL, instead of 200 µL, was used. The DNA concentration was quantified in each sample with NanoDropPhotometer (Thermo Fisher Scientific), and DNA quality was assessed with the use of the 260 nm/280 nm absorbance ratio. Genotypes of CYP2C9, CYP2C19, CYP2D6, CYP3A5, DPYD, SLCO1B1, TPMT and VKORC1 were determined using the Drug Metabolizing and Transporters (DMET) Plus Array (Affymetrix, Santa Clara, CA). CYP2D6 copy number variants were detected with qPCR (Thermo Fisher Scientific, Massachusetts, USA). The DMET array was supplemented with the DPYD 1236G>A and 2846A>T variants which were routinely tested in clinic at the LUMC. Validation of the assays is described elsewhere [29 ].