Massarray assay design version 3

Genomic DNA from white blood cells of probands and their family members was used for genotyping MLH1 (NM_000249.3), APEX1 (NM_001641.3), MUTYH (NM_012222.2), OGG1 (NM_016828.2), NUDT1 (NM_002452.3), XRCC5 (NM_021141.3), XPA (NM_000380.3), and ERCC2 (NM_000400.3) (Table 1). Genotyping was performed using Sequenom iPLEX MassARRAY (Sequenom, Inc., San Diego, CA, USA). Matrix‐assisted laser desorption ionization time of flight (MALDI‐TOF) spectroscopy was performed using the Sequenom MassARRAY platform and iPLEX GOLD chemistry as described in our previous studies (Kamiza et al., 2015, 2016, 2018). We added 10 ng of template DNA in polymerase chain reaction (PCR) mixture containing Qiagen HotStarTaq. We conducted primer extensions and shrimp alkaline phosphatase by using guidelines from Sequenom. Primers used for PCR were from Integrated DNA Technologies (OH, USA). Assays were designed using MassARRAY Assay Design, Version 3.1 (Sequenom). We repeated 10% of randomly selected samples for quality control and results showed 100% concordance for all the SNPs.

DNA was extracted from white blood cells by using the standard procedures of phenol and chloroform extraction. TP53 rs1042522 and rs12947788 were genotyped using the Sequenom iPLEX MassArray (Sequenom, Inc., San Diego, CA). We performed iPLEX genotyping through MALDI-TOF spectroscopy by using the Sequenom MassARRAY platform and iPLEX GOLD chemistry. We used 10 ng of genomic DNA as a template and added a PCR mix containing Qiagen HotStarTaq to the template. Shrimp alkaline phosphatase and primer extension steps were performed using the Sequenom protocol and reagents. Primers were obtained from Integrated DNA Technologies (OH, USA). Assays were designed using the MassARRAY Assay Design, Version 3.1 (Sequenom). Raw genotype data were visualized and processed using the MassARRAY Typer software, Version 4.0. Genotyping was repeated on 10% of the samples for quality control.