Genomic DNA was obtained using the Easy-DNA kit (Invitrogen) following the manufacturer's instructions. The genotype of each sample was obtained by polymerase chain reaction (PCR)-restriction fragment length polymorphism, according to the method originally described by Senol Tuncay et al[12 (link)] with a few modifications. Every reaction was performed with 0.5 μM of each primer –forward: 5′-TGG GCA CAA GTC GTT TAT GA-3′ and reverse: 5′-CTG GAG CCG GTA GGG AAG-3′– 0.5 mM of dNTPs, 2 mM of MgCl2, 2.5 μL of PCR 10× buffer, 2 U of Taq polymerase (Invitrogen), and 200 ng of DNA, in a final volume of 25 μL. The thermocycler program was set at 30 seconds at 98°C, 40 cycles of 5 seconds at 98°C, 5 seconds at 65°C, and 5 seconds at 72°C, followed by a final extension of 5 minutes at 72°C. The expected size of the PCR product was 285/280 bp for the Ins/Del alleles.
The polymorphism was detected using 5 μL of the PCR product, digested with 3 U of the PfIMI (Takara) enzyme and left overnight at 37°C. The digestion products were observed in a 2% agarose gel. The Ins allele (exscinded by PfIMI) appeared at 45 and 245 bp, the Del allele at 280 bp.
The polymorphism was detected using 5 μL of the PCR product, digested with 3 U of the PfIMI (Takara) enzyme and left overnight at 37°C. The digestion products were observed in a 2% agarose gel. The Ins allele (exscinded by PfIMI) appeared at 45 and 245 bp, the Del allele at 280 bp.