PCR-based identification of short deletion/insertions and single nucleotide substitutions in genotyping of splotch (Pax3sp) and truncate (Nototc) mouse mutants

Splotch (Pax3sp) and truncate (Nototc) are spontaneously arisen mouse mutants with disturbed embryo development. Splotch carries a Pax3 mutation and it is characterized by the neural tube defect. Corresponding mutation in human causes Waardenburg syndrome. Truncate is Noto mutant with disturbed development of the caudal notochord. In order to establish easy genotyping procedure of these mutations, it was tested whether simple PCRs with single primer pairs could be used for this purpose. As it was necessary to differentiate sequence variants on the scale of one to several nucleotides, the approach referred to as “3′ variable primer ends” was applied. The method was based on the presence of discriminating nucleotides at the 3′ end of the primer sequence. This approach was successfully applied in genotyping adult mice and embryos of splotch with a 6 bp deletion/insertion and truncate with a single nucleotide substitution. Described genotyping approach facilitates recognizing of these mutations and it could be in general used for detection of sequence differences in one to several nucleotides.