Mutant analysis and the building of complex strains bearing several mutant alleles and/or transgenes are precious tools routinely used in studies using genetic models such as C. elegans. Crossing, strain building and genotyping necessitate to follow the various alleles to be combined during the crosses, some of which may not exhibit any easily detectable phenotype. Phenotypic markers (usually associated with an obvious phenotype: e.g. dpy, unc, rol, lon, etc) have been used as balancers in trans to follow a mutation of interest. This strategy is best used when the marker gene is located close enough to the mutation that one wishes to follow, to avoid crossing overs and recombinations during the several crosses and homozygosing steps. In recent years, using MosSCI insertions, Christian Frokjr-Jensen et al. have generated a set of strains bearing fluorescent markers inserted at specific chromosomal locations that can also be used as dominant genetic markers (Frokjaer-Jensen et al. 2014).However, depending on the location of the mutation of interest on a chromosome, there might not be any close genetic marker that can be used. In addition, the use of genetic markers to follow a mutation of interest imposes to first build one or several intermediate strains to associate two or more mutant alleles. To introduce a chromosome-integrated transgene in the background of a mutant, strategies using genetic markers can also be limited: the phenotypes of different genetic markers are not all compatible, and the fluorescent expression in MosSCI strains can preclude detection of other transgenes needed in the strain of interest. Finally, other species, from other worms to vertebrates, may not have the same tools such as the extensive C. elegans genetic markers and mutant strains library.Here, we describe a simple and quick method to follow mutations through the several crossing steps. This method is particularly handy when the mutation of interest does not create or modify a restriction site, and avoids having to resort to a multi-step process involving the individual sequencing of multiple animals, after amplification of the genomic region of interest. It is based on the creation of a restriction site by PCR specifically in the amplicon for one of the alleles only, using specific oligonucleotides to amplify a small DNA fragment around the mutation to be followed, and can be applied to several mutations in parallel. This method which uses only two primers for a given gene, one round of PCR and just one PCR product for each allele is also simpler and more robust than the tetra primer ARMS-PCR previously described (Ye et al.. 2001).To date, we have applied this method to the genotyping of 116 mutant alleles, including
daf-2(
e1370),
ceh-6(
gk665),
sem-4(
n1971,
n2654