We recently had a project in which we needed to cross 3 mutant C. elegans strains to obtain a worm that was homozygous at all three loci. Two loci could be easily genotyped using traditional allele-specific PCR (ASPCR) but the third locus was a single nucleotide polymorphism (SNP). SNPs can be very difficult to genotype because they only have one base pair difference from wild-type. The only published method to identify this SNP used the observation of the phenotype caused by the point mutation. This point mutation results in the feminization of the C. elegans gonad, meaning that the strain could not self-fertilize when the mutation is homozygous and requires the strain to be maintained using mating. To genotype this strain we would need to wait until the worms reached maturity to identify adult worms that lacked sperm or primary spermatocytes, while still working fast enough to identify worms that could still be mated. Using a phenotype to confirm a strain is laborious and time consuming with a lot of false positives and negatives. We were also unable to determine heterozygosity in one generation, which added significantly to the timeline and frustrations of the project.
Since this phenotype is difficult to identify, to maintain as a heterozygote and to use to confirm the third locus was homozygous, we set out to find another molecular method that would help us confidently genotype this locus. The molecular methods our lab has successfully used in the past to identify SNPs are restriction fragment length polymorphism (RFLP), allele-specific (AS) primers, high resolution melting analysis (HRMA) and sequencing. All of which failed to discriminate between the mutant and wildtype alleles or were not fast enough to effectively identify worms still young enough to mate. Additional molecular methods (e.g. array-based hybridization, TaqMan probes, etc) are available. However, they tend to be optimized for large populations or whole genome studies and can be time consuming and cost prohibitive.
Luckily for me my colleague had read a paper about a new robust genotyping method that was published last year. “Cost-effective and robust genotyping using double-mismatch allele-specific quantitative PCR “( Scientific Reports, 9(1), 2150) describes a method that seemed appropriate and promising. If it worked it would allow us to identify a single base pair mismatch in a cost effective way on a single worm lysate. It seemed too good to be true and I went into testing the method with a heavy amount of skepticism.
Double-mismatch allele-specific (DMAS) quantitative PCR successfully genotyped our SNP with ease. We were able to use reagents and equipment that we already had available for qPCR and HRMA screens. Once implemented, we rapidly identified heterozygous animals allowing us to mate offspring immediately upon maturity. This allowed us to greatly reduce the amount of worms isolated and decreased the turn around time of genotyping. Using this new genotyping technique, we were able to identify and maintain a triple loci homologous mating within a month of its implementation.
Two major lessons I learned from this experience is to stay current with a broad variety of literature and to always be willing to try new methods. Both of these tasks may seem overwhelming but in the end it can save you months of frustration at the bench.
- Genetics 119: 43-61 (May, 1988).
- Lefever, S., Rihani, A., Van der Meulen, J., Pattyn, F., Van Maerken, T., Van Dorpe, J., et al. (2019). Cost-effective and robust genotyping using double-mismatch allele-specific quantitative PCR. Scientific Reports, 9(1), 2150)