4.3. T7E1 Assay

Genomic DNA was extracted from transfected pool of cells as mentioned earlier, and the target gene region in OCRL (Exon 3) was amplified using OCRL specific primers (Forward Primer: 5′-CACCACTAGCATCCTTTTAGGC-3′ and Reverse Primer: 5′-AGAGAAAGTATCATCTCCTCAATGT-3′). T7 Endonuclease I (M0302S, New England Biolabs, Hitchin, UK) was used to digest PCR products, which were denatured and annealed to facilitate the formation of heteroduplex DNA. The enzyme was incubated with PCR product for 90 min at 37 °C. The reaction was stopped by adding 0.25 M EDTA and the digested products were separated using 2% agarose gel electrophoresis to check for fragmented DNA.