How does Guide-it Flapase detect the edited nucleotide?
How does Guide-it Flapase detect the edited nucleotide?
Guide-it Flapase is a recombinant, structure-specific nuclease from Takara Bio; it recognizes and cleaves double-flap structures, but is unable to cleave gapped structures. The Guide-it SNP Screening Kit employs this feature to assay for single-nucleotide substitutions: the main difference between the double-flap and the gapped structures is whether or not hybridization occurs between the flap-probe oligo and the PCR product at the position of the interrogated base.
In a scenario where the interrogated base is edited, the flap-probe oligo containing the complementary nucleotide forms a complete base pairing at the target site. If the editing event is unsuccessful and the interrogated base is wild-type, there isn't a complete base pairing at the target site, and a gap is formed.