Optimization of ITS rDNA Amplification for Fungi from Black Soil in North China

Abstract

In order to optimize polymerase chain reaction (PCR) amplification of the internal transcribed spacer (ITS) rDNA region from fungi found in black soil in North China, an orthogonal experimental design [L16(45)] was used to evaluate five factors (template, Mg2+, dNTP, Taq DNA polymerase, and primer) from four levels. Subsequently, the optimal annealing temperature, annealing time, extension time and cycle numbers were evaluated. The results showed that the optimized PCR solution for amplification of ITS region comprised 5 μL 10× buffer, 30 ng soil DNA template, 3.0 mmol · L−1 Mg2+, 0.2 mmol · L−1 dNTPs, 0.1 μmol · L−1 each forward and reverse primer, and 2.0 U Taq enzyme in 50 μL reaction volume. The optimal thermal cycling protocol consisted of initial melting at 94°C for 5 min, followed by 35 cycles at 94°C for 30 s, 56°C for 30 s, 72°C for 90 s, and a final extension of 72°C for 10 min.