Gel-free genotyping of deletion alleles in Caenorhabditis elegans with real-time PCR

C. elegans is a powerful model system for studying the genetic basis of biological processes. Identifying relevant genes and organizing them into genetic pathways often involve analyzing phenotypes when mutations for two or more genes are combined in the same strain. This requires crossing and genotyping resulting individual F2 lineages. In the simplest case, mutant alleles cause robust phenotypes that can be observed with a microscope. Alleles without easily observable phenotypes can be followed by including closely linked alleles with obvious phenotypes (Wang and Sherwood 2011; Fay 2013). In some cases, neither of these scenarios are possible, which leaves the mutated allele as the only factor for selection. The traditional method of genotyping deletion alleles in C. elegans involves PCR amplification and detection of distinct product sizes after electrophoresis (Liu et al. 1999; Ahringer 2006). This method is reliable and permits visualization of product lengths, but requires additional steps to cast, load, run and visualize gels after PCR. Laboratories that study gene expression commonly use real-time PCR systems for quantitative RT-PCR. Here, we tested if real-time PCR could also be used to screen worm DNA lysates for deletion alleles. Real-time PCR systems detect accumulation of products in each well during cycling using double-stranded specific DNA dyes and arrays of illuminators and photodetectors; amplification of product can be viewed during cycling. Cycle threshold (Ct) is calculated automatically by real-time PCR systems as the earliest cycle that fluorescence exceeds background and is inversely related to log of starting template concentration. After the last PCR cycle, fluorescence is monitored in each well over a range of temperatures to produce a melting curve that can help distinguish different amplicons. In theory, real-time PCR should be able to detect the 2:1 ratio in template DNA concentration between homozygous and heterozygous worms. However, a quantitative approach to identify heterozygotes would require biological replication and amplification of a separate normalizing gene to be reliable (Radonić et al. 2004; Zhang et al. 2012). We instead devised a qualitative approach where wild type and deletion alleles are detected for each sample in separate DNA-dye reactions run together on the same plate in a real-time PCR machine. Wild type ‘inner’ primers amplify a portion of the sequence within the deletion and mutant ‘outer’ primers amp