Since these data were based on whole genome sequencing that could overlook specific mutations, we performed targeted sequencing in the tumor (TCGA-B6-A1KF) with very high levels of 2-HG to look for the recurrent mutations found in other tumors in IDH1 and IDH2.
Using this hybrid protocol, we targeted mutations to over 30 sites within an approximately 700 bp region.
As a final test of the effect of the distance of the mutation to the marker insertion site, we targeted a mutation 1.0 kb from the insertion site using a 1.1 kb PCR fragment.
To test this and model the role of the Int6 mutation in alveolar hyperplasia, we targeted Int6sh expression to the differentiating alveolar epithelium using the whey acidic protein (Wap) promoter, which is under tight hormonal regulation in the mouse mammary gland [ 13, 14].
To confirm the EMS-induced mutation, we used targeted Sanger sequencing (Purdue Genomics Core facility) of PCR products from DNA isolated from dhr2-1 mutands and BTx623 (data not shown).
We targeted the mutations to the upstream half of RPB2 because the N-terminal portion of the Rbp2 subunit contains several regions of high sequence and structural similarity shown to be important for termination in other RNAPs, as well as fairly extensive regions that are conserved in but unique to eukaryotic Pol II enzymes (Sweetser et al. 1987).
Alignment of these regions from ch-TOG orthologs highlighted a conserved patch including a pair of leucines which we targeted for mutation (Fig. 4B).
We targeted this new mutation in the 5′ end of the DOCS1 cDNA (Fig. 1).
"What's hugely encouraging is that many of the key mutations we have identified are ones targeted by existing cancer drugs, meaning that we could be entering a new era of personalised cancer treatment," he said.
As a second method to evaluate the likelihood of off-target mutations, we performed local alignments of our guide sequences to all Cas9 target sequences whose PAM site was within 30 nucleotides upstream and downstream of a detected variant, as well as to 10,000 randomly selected Cas9 target sequences from the genome.
