Figure 3—figure supplement 2.

Figure 3—figure supplement 2.

(A) 5’–3’ genomic region of the wild-type and mutated alleles of the stable mutant lines we used to test the detection of small deletions by headloop PCR. apoea: 1 bp deletion followed by a T > A transversion. cd2ap: 2 bp deletion. Framed: headloop tag; grey underlay: headloop tag binding site. (B) For each mutant line (apoea, cd2ap), 32 embryos issued from a heterozygous to wild-type outcross were genotyped (around 50% of embryos expected to be heterozygous and the rest wild-type). Bands on agarose gels alternate between standard PCR (std) and headloop PCR (HL). The genotype (wild-type or heterozygous) was called based on the presence or absence of the headloop PCR band. If the embryo was wild-type, headloop PCR did not generate a product. If the embryo was heterozygous, headloop PCR generated a product, as half of the template DNA was mutated. (C) All genotype calls by headloop PCR were then verified by Sanger sequencing. Samples traces are included here. For the standard PCR heterozygous samples, two Sanger traces run alongside each other past the mutation site, as templates from both the wild-type and the heterozygous alleles were sequenced. Conversely, the Sanger trace for the headloop PCR heterozygous samples remains unique past the mutation as only the mutated allele was sequenced, which confirms that the wild-type allele was not amplified. (D) Summary of genotyping results by headloop PCR and Sanger sequencing for the two clutches of 32 embryos.