Metazoan genomes contain thousands of sequence tracts that match the guanine-quadruplex (G4) DNA signature G3NxG3NxG3NxG3, a motif that is intrinsically mutagenic, probably because it can form secondary structures during DNA replication. We and others have previously shown that in C. elegans, the gene dog-1 is required to prevent deletion formation at these G4 sites (Chueng et al, Kruisselbrink et al). Here we provide a protocol to show how this feature can be used to isolate deletion alleles of many Caenorhabditis elegans genes.
Consult Wormbase or the complete list of G4 DNA sites (available as Supplemental Table with Pontier et al, 2009) whether your gene of interest is close (preferably within 2 kb) to a G4 DNA site.
When designing primers, take into account that G4 DNA-induced deletions always occur in one particular orientation: the deleted DNA is always upstream of the G4 sequence. Thus, primers are chosen such that one primer is close to the 3’ side of the G4 tract, whereas the other primer is 1 kb upstream (Figure 1). Nested PCRs increase sensitivity, and we had the best results with product sizes of 1200 bp for the (wild-type) outer product, and ~ 1000 bp for the inner product. Primers can be chosen further apart, but this may hamper detection of smaller deletions.