A MassARRAY3 Analyzer (Sequenom Inc, San Diego, CA) with iPLEX Gold Genotyping Reagent (Sequenom Inc) was used to validate the 286 candidates, according to the manufacturer's instructions. Briefly, MassARRAY Typer4 Assay Designer (Sequenom Inc) designed the 286 PCR primer pairs and 286 iPLEX primers as single-base extension primers for each candidate. We used 37 genomic DNA samples, including 35 samples from the TOY-KO pedigree and two control samples, as well as C57BL/6J and the original ES cell DNA to determine the origin of the de novo mutations in the TOY-KO pedigree. Ten nanograms of genomic DNA were used in each multiplex PCR for the MassARRAY. After dephosphorylation, single-base extension with the iPLEX primer and desalting were performed. The reaction products were spotted onto a 384-format SpectroCHIP with a MassARRAY Nanodispenser (Sequenom Inc) and then subjected to a MassARRAY 3 analyzer (Sequenom Inc). MassARRAY Typer 4.0 software (Sequenom Inc) was used to analyze the mass spectrum data.
Ohno M., Sakumi K., Fukumura R., Furuichi M., Iwasaki Y., Hokama M., Ikemura T., Tsuzuki T., Gondo Y, & Nakabeppu Y. (2014). 8-oxoguanine causes spontaneous de novo germline mutations in mice. Scientific Reports, 4, 4689.
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