Fig. 2

Genotype-specific PCR primers compete for targets to increase amplification specificity. Fluorescently labeled genotype-specific forward primers for MBD3 were used to pair with a common reverse primer individually or in combination in PCR reactions to amplify the gene-editing target region of MBD3 in genomic DNA isolated from wild type (Wt), heterozygous mutant (Het), and homozygous mutant (Hom) animals, respectively. The PCR products were resolved on a gel and scanned to visualize the fluorescent signals as in Fig. 1. Note that the wild type-specific forward primer (Fwt) non-specifically amplified the target region in the homozygous mutant template to produce a strong band when paired alone with the common reverse primer (circled in lane 4). However, the band was absent when both wild type-specific forward primer and mutant-specific forward primer were present together in a single tube dual fluorescent PCR reaction (circled in lane 10), likely due to more effective competition of the mutant-specific forward primer to bind to the mutant templates for PCR amplification. Similarly, the weak non-specific amplification of mutant-specific primer on the wild type template (circled in lane 5) when wild type-specific forward primer was absent was also inhibited in the dual fluorescent PCR reaction when the wild type-specific forward primer was also present (circled in lane 8). Ctrl: control PCR of DNA template from heterozygous animals as reference to adjust green and red signals for visualization