Figure 4. Analysis of putative analog sensitive clones using the second generation repair template via PCR. (a.) A schematic of the sequence and putative splicing pattern resulting from the use of the second generation repair template. Nucleotides modified in the second generation are underlined (see text). Note the suppression of mis-splicing via elimination of the splice site acceptor (AG) by the silent A->T mutation (grey arrow). (b.) A schematic of CDK12 exon 6 with the positions of the homologous repair template (blue box), the analog sensitive mutation (red star), and the PCR primers specific to the analog sensitive (AS) and wild type (WT) sequences indicated; the PCR product is expected to be 560 bp. (c.) PCR of genomic DNA from 7 putative analog sensitive clones is visualized using a 1.5% agarose gel stained with EtBr; the top section of the gel displays the results using AS specific primers while the bottom section of the gel displays the results using the WT primers. Due to the changes in the repair template the new AS specific forward primer is not as specific as in Fig. 1, and fires at a low rate even on WT templates (lanes 2 and 4-8). Note clone #2, which appears to be homozygous for the analog sensitive mutation.