Premix hot start version

To determine the effective annealing temperature for the LNA oligonucleotides, PCR amplification was performed at different annealing temperatures for the bacterial primer by using a PCR thermal cycler (PC350 ASTEC, Fukuoka, Japan). DNA extracted from rice and wheat roots was used for the amplification. The PCR mixture contained 12.5 μL of Premix Hot Start Version (Takara, Otsu, Japan), 1.0 μL of each primer (20 pmol μL⁻¹), and 1.0 μL of the DNA template. Sterilized ultrapure water was added to a total volume of 25 μL. The amplification conditions were as follows: 94°C for 3 min (initial denaturation), followed by 30 cycles at 94°C for 1 min, annealing from 60°C to 72°C (2°C increments) for 1 min, and 72°C for 2 min, with a final extension step at 72°C for 10 min. After the amplification, aliquots of the PCR products were electrophoresed on a 1.5% agarose gel, and the effective annealing temperature of the LNA oligonucleotides was visually estimated from the product intensities.

In order to estimate the Tm values of LNA oligonucleotides, PCR amplification was performed at different annealing temperatures using a PCR thermal cycler (PC350, ASTEC, Fukuoka, Japan). DNA extracted from wheat and soybean roots was used as representative samples. The PCR mixture contained Premix Hot Start Version (Takara, Otsu, Japan), the fungal primer set (0.8 μM each), and the DNA template. PCR conditions were as follows: 94°C for 3 min (initial denaturation), followed by 40 cycles at 94°C for 1 min, annealing from 60°C to 74°C at 2°C intervals for 1 min, and 72°C for 2 min, with a final extension step at 72°C for 10 min. Aliquots of the PCR products were electrophoresed after amplification. The Tm values of the LNA oligonucleotides were estimated from amplicon intensities.