Fig. 1

Co-PCR with two genotype-specific fluorescent primers faithfully distinguishes the three genotypes of a genome-editing target gene. Primers labelled with fluorescent IR700 and IR800 dyes that were specific for the wild type and mutant alleles of indicated target gene, respectively, were mixed together with a third primer common to both wild type and mutant alleles in single tube for competitive PCR to genotype animals with mutation in HAL2 A and MBD3 B. The PCR products were denatured and resolved on 15% Urea-PAGE gels. Fluorescent bands were digitally visualized on a LI-COR Odyssey Clx Scanner with the IR700 signal recorded as green and IR800 signal as red. The fluorescent densities were adjusted in each individual channel to the condition where the green and red fluorescent densities on PCR products of heterozygous mutant targets (Het) were about equal, which generated a yellow band in the merged field. The wild type (Wt) and homozygous mutant (Hom) bands were red and green, respectively. Note that genotype-specific primers could be either reverse primers (Rm and Rwt in panel A) or forward primers (Fm and Fwt in panel B), and should target the same region with only a short stretch of sequences different at the 3’-end (in purple letters). The arrows point to the PCR products and the star * indicates the unincorporated primers