Figure 1. CEPAD-PCR method overview and screening: (A, A') Schematic of ARMS-PCR (A) and CEPAD-PCR (A') with an expected gel electrophoresis pattern common to both methods. Internal primers which bind to wildtype (pink) or the edited mutant (green) are shown. Brown arrows mark the external primers. Different colors on the gel (right) indicate the specific mutant or wildtype bands with colors being represented by primer pairs. Black nucleotides indicate 'unchanged' nucleotides, cyan indicate silent mutations, and orange indicate CRISPR-Cas9 edited mutations. Light green stripe indicates a mismatch only present in the primer. (B) CRISPR-Cas9 edit scheme of
raga-1(Q63L) mutation with edited (orange) and silent (blue) mutations for CEPAD-PCR. Internal primers for CEPAD-PCR are shown overlapping over the mutated region, the 3' end of which is on a mutated nucleotide for each. (C) F1 worms screened with CEPAD- PCR. Lanes with an additional band corresponding to the
raga-1(Q63L) mutant allele are labeled (asterisk). (D) F2 worms screened with CEPAD-PCR. Shown are representative banding patterns for wildtype and
raga-1(Q63L), heterozygous and homozygous. (E) Sequencing results for one isolated line. (F) CRISPR-Cas9 edited
daf-15(S907AS948A) allele with silent mutations for removal of a PAM site (blue). Wildtype (reverse, magenta) and mutant (forward, green) primers overlap by 3 nucleotides, two of which are silent mutations. (G) CEPAD-PCR screening for
daf-15(S907AS948A). A faint ghost band corresponding to the wildtype size is present in the homozygous mutant.