Sephadex g 50 superfine columns

Each polymorphic region was amplified by PCR using the same reaction mixture and conditions described in the ‘Genotyping’ section above. The PCR product (5 µL) was reacted with Exonuclease I (Epicentre, Madison, WI, USA) and shrimp alkaline phosphatase (Affymetrix, Inc., CA, USA) to inactivate the PCR primers. Cycle sequencing reactions were performed using the BigDye Terminator cycle sequencing kit (version 3.1; Life Technologies) according to the manufacturer’s protocol. The reaction solution consisted of 25 ng of template DNA, sequencing buffer, 0.1 µM of forward primer or 0.1 µM of reverse primer, and distilled water (total reaction volume, 10 µL). The reaction was carried out with a 30-sec hot start at 96 °C, followed by 25 cycles of 10 s at 96 °C, 5 s at 50 °C, and 4 min at 60 °C, with a final elongation step at 60 °C for 4 min. The reaction solution was purified using Sephadex G-50 superfine columns (GE Healthcare, Chicago, IL, USA) and dried before adding 15 µL of Hi-Di formamide (Life Technologies). The DNA was denatured at 95 °C for 2 min, incubated on ice for at least 5 min, and subjected to capillary electrophoresis on an ABI PRISM 3130xl instrument (Life Technologies) to determine the DNA sequence (Table 2).

The respective polymorphic regions were amplified using PCR performed with the same reaction fluid composition and conditions as the PCR-RFLP method. After the PCR, to deactivate the dNTPs and PCR primer, Exonuclease I (Epicentre, Edgewood, MD, USA) and Shrimp Alkaline Phosphatase (Affymetrix, Inc., Sunnyvale, CA, USA) were added to 5 µL of PCR products and enzymatically reacted for 20 min at 37 °C. Then, template DNA was prepared via deactivation for 20 min at 80 °C. Subsequently, the cycle-sequencing reaction was performed according to the protocol of the BigDye^® Terminator v3.1 Cycle Sequencing Kit (Life Technologies). Distilled water was added to 25 ng of template DNA, sequencing buffer, and forward or reverse primer (0.1 μM) to attain a total reaction fluid of 10 µL. The hot-start reaction was performed for 30 s at 96 °C; after 25 cycles (10 s at 96 °C, 5 s at 50 °C, and 4 min at 60 °C), elongation was performed for 4 min at 60 °C. The reaction solution was refined using Sephadex G-50 superfine columns (GE Healthcare, Chicago, IL, USA) and dried, followed by the addition of 15 μL Hi-Di formamide (Life Technologies). After denaturation for 2 min at 95 °C, the mixture was placed on ice for ˃5 min, and capillary electrophoresis was performed using an ABI PRISM 3130xl (Life Technologies) to determine the DNA base sequence.