Conventional PCR

Conventional PCR was performed on 2 μL of extracted DNA to amplify a 500 bp region as previously described (10). Briefly, 3 μL each of forward (5′-CAA GGT ATC GAT CGT CTC TCT ACT −3′) and reverse (5′-TGA GGG TAG TCT TGC ACG CGA AT-3′) primers were used at 10 μM concentrations in a total reaction volume of 50 μL containing AmpliTaq Gold® DNA Polymerase (Applied Biosystems) and 25 mM magnesium chloride (Promega) in GeneAmp assay buffer II (Applied Biosystems). Reactions were carried out using a 9,600 GeneAmp PCR system (Perkin Elmer, Waltham, MA) with the following conditions: 95°C 4 min; 40 cycles of 94°C 1 min, 63°C 1 min, 72°C 30 s, and a cycle of 94°C 1 min, 63°C 1 min, 72°C 10 min. The reaction product (20 μL) was visualized on a 1.5% agarose gel containing 0.5 mg ethidium bromide per mL of agarose solution.