Pyromark q96 system

To determine changes in frequency of key DNA mutations linked with SDHI resistance at each round of selection, six populations: three exposed to UV and with a greater diversity of mutations (I+1, H+1, and H+2), and the others without UV exposure and with fewer or no mutations (L−1, I−1, and H+1) were tested using SNP detection pyrosequencing assays using the PyroMark Q96 system (Biotage, Uppsala, Sweden) as described by Carter, Cools, West, Shaw, and Fraaije (2013) (see Supporting information). Aliquots of 10 μl of spore suspension from the last round of selection, and stored glycerol spore suspensions of previous RS, were plated out on YPD agar, and incubated for seven days at 15°C in the dark. DNA was extracted from each population. Mutants carrying different Sdh variants and the reference isolate IPO323 were used as positive and negative controls for the SNP detection pyrosequencing assays. Frequencies of mutations underlying SdhB‐H267L, B‐H267Y, B‐N225T, C‐T79I, C‐S83G, C‐H152R, and D‐I50L were estimated with the PyroMark Q96 ID software version 2.5. Frequency values are the mean of two technical replicate pyrosequencing reactions. The detection limit of SNP detection pyrosequencing is approximately 3% for each allele (Gruber, Colligan, & Wolford, 2002; Wasson, Skolnick, Love‐Gregory, & Permutt, 2002).

An epigenetic analysis was performed by bisulfite pyrosequencing, which is a sensitive and accurate method for the detection of methylation. DNA was treated with sodium bisulfite using the EpiTect Bisulfite Kit (Qiagen), which was followed by the cleanup of bisulfite-converted DNA. PCR amplification was then performed with a PyroMark CpG Assay Kit (Qiagen) and a PyroMark Gold Q96 Reagent Kit (Qiagen) in a PyroMark Q96 system (Biotage AB, Uppsala Sweden). PyroMark PCR master mix includes HotStarTaq DNA polymerase and optimized PyroMark reaction buffer that contains 3 mM MgCl2 and dNTPs, 10x CoralLoad Concentrate, 5x Q-Solution, 25 mM MgCl2, and RNase-free water. The PCR amplicon covers the sequence of human chomosome 3q: NC_000003.12 (179148114.179240084). Nine CpG sites are located in the promoter region of the PIK3CA gene (-100 bps) as indicated with the bold letter ‘C’ and recorded as CpG1-9 (Figure 6A). Finally, the methylation levels of these CpG sites were detected with a PyroMark Gold 96 Reagent Kit (Qiagen) and a PyroMark Q96 ID pyrosequencing system (Biotage). The unmethylated and unconverted DNA samples (Qiagen) were used to control the conversion efficiency during the bisulfite treatment and the accuracy of the methylation analyses. PyroQ-CpG software (Biotage) was used for methylation data analysis.

Epigenetic analysis was performed by bisulfite pyrosequencing, which is a sensitive and accurate protocol [34 (link),35 (link)]. DNA was treated with sodium bisulfite using EpiTect Bisulfite kit (Qiagen) and cleanup of bisulfite-converted DNA was done. PCR amplification was then carried out using PyroMark CpG assay (Qiagen) and PyroMark Gold Q96 Reagent kits (Qiagen) in a PyroMark Q96 system (Biotage AB, Uppsala Sweden). PyroMark PCR master mix includes HotStarTaq DNA polymerase and optimized PyroMark reaction buffer containing 3 mM MgCl² and dNTPs, 10x CoralLoad Concentrate, 5x Q-Solution, 25 mM MgCl2, and RNase-free water. The PCR amplicon covers the sequence in human chromosome 8: 117962434 to 117962479 (version 37.56). There are four CpG sites in the SLC30A8 gene promoter region as indicated with the bold letter ‘C’ and recorded as CpG1-4 (Figure 1). Finally, methylation levels of these CpG sites were detected by using the PyroMark Gold 96 Reagent kit (Qiagen) and a PyroMark Q96 ID pyrosequencing system (Biotage). The unmethylated and unconverted DNA samples (Qiagen) were used for control of conversion efficiency in bisulfite treatment and accuracy in methylation analyses. PyroQ-CpG software (Biotage) was used for methylation data analysis.