Measuring Targeting Efficiency
A widely used method to identify mutations is the T7 Endonuclease I mutation detection assay (1,2). This assay detects heteroduplex DNA that results from the annealing of a DNA strand, including desired mutations, with a wild-type DNA strand. The EnGen Mutation Detection Kit provides optimized reagents for performing robust T7 Endonuclease-based detection of genome editing events.
Reyon, D. et al. (2012) Nat. Biotechnol., 30(5):460-5
Vouillot, L., et al. (2015) G3 (Bethesda), 5(3):407-15
Genomic DNA is amplified with primers bracketing the modified locus. PCR products are then denatured and re-annealed yielding three classes of possible structures. Duplexes containing a mismatch greater than one base are digested by T7 Endonuclease I. The DNA is then electrophoretically separated and fragment analysis is used to estimate targeting efficiency.