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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 Frokjær-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 & n1378), mab-9(e2410), eri-1(mg366), egl-5(n945), egr-1(ku285), jmjd-3.1(fp15), wdr-5.1(ok1417). The results for mab-9(e2410) are shown in figure 1 and have been repeated 5 times. mab-9 is T-box transcription factor well studied for its role in hindgut and tail development. Unfortunately, this gene is in a genomic region poor in phenotypic balancer genes, and shows a subtle mutant phenotype under dissecting microscope (https://wormbase.org/species/c_elegans/gene/WBGene00003106#0-9f-3). The mab-9(e2410) allele exhibits two point mutations (TA to GT) in the 4th intron, that cause a splice defect (Woolhard & Hodgkin 2000, Figure 1A). The WT sequence is gtaact and the mab-9(e2410) sequence gGTact. To follow this mutant allele during crosses, it is possible to modify one nucleotide by PCR and specifically create an Acc65I site (G/GTACC) in the amplicon from the mutant allele: By replacing in the mab9R reverse primer the last T of the mutant gGTact sequence by a C, an Acc65I gGTacc site is created in the mutant amplicon only (Figure 1A). Primers mab9F and mab9R allow amplification of a 358 bp PCR fragment. After Acc65I digestion, the PCR product from mab-9(e2410) is digested into a 279bp and a 79bp fragments, while the WT version remains 358pb, and heterozygous animals exhibit three bands at 358bp, 279bp, and 79bp (Figure 1B). Using this method, genotyping is achieved in a couple of hours enabling to pick the desired animals the same day. Since it bypasses the need to build genetically balanced strains, the time to obtain the final strain is considerably reduced. This PCR-based strategy is widely adaptable to many other organisms than C. elegans, from well-known genetic models such as drosophila, zebrafish or other worms to models lacking a full genetic tool box. |
