Genomic DNA Extraction and Mutation Detection

To detect mutations at the target site, genomic DNA was extracted from G₁ neonate larvae or a leg of an adult moth using the hot sodium hydroxide and tris (HotSHOT) method [79 (link)]. Genomic DNA was also prepared from a third instar larva used in total RNA extraction described below using TRIzol reagent (Invitrogen), according with the manufacturer’s protocol [80 (link)]. Genomic PCR was conducted using KOD One (TOYOBO) with the primer sets listed in S6 Table under the following conditions: 40 cycles of denaturation at 98°C for 10 s, annealing at 60°C for 5 s, and extension at 68°C for 5 s. The PCR product was annealed under the following conditions: 95°C for 10 min, 85°C for 1 min, 75°C for 1 min, 65°C for 1 min, 55°C for 1 min, 45°C for 1 min, 35°C for 1 min, and 25°C for 1 min, followed by incubation at 4°C. The annealed PCR products were cleaved by T7ENI (NEB) at 37°C for 1 h. The cleavage products were detected by agarose gel electrophoresis. For the heteroduplex mobility assay, the annealed PCR products were electrophoresed using the MultiNA microchip electrophoresis system (SHIMADZU) with the DNA-500 reagent kit [81 (link),82 (link)].