Abstract

Optimization of SCoT-PCR reaction system in Dactylis glomerata by orthogonal design

Author(s): B. Zeng, X. Huang, L.K. Huang, J. Zhang, H.D. Yan, D. Luo, H. Liang and Y. Yuan

The effects of 5 factors (template DNA, Mg2+, dNTPs, Taq DNA polymerase, and primer) on the polymerase chain reaction (PCR) were investigated to optimize the start codon targeted polymorphism (SCoT)-PCR system of Dactylis glomerata L., using an orthogonal design L16 (45). A suitable SCoT-PCR system for D. glomerata was established; the 20 μL reaction volume contained 3.0 mM Mg2+, 0.2 mM dNTPs, 1.0 U Taq DNA polymerase, 0.2 μM primer, 20 ng template DNA, and 2 μL 10X buffer. Each factor had a different effect on the amplification reaction, and the concentration of dNTPs had the largest effect on the SCoT-PCR system. We tested 10 orchardgrass samples to determine and verify the stability of the reaction system. The results showed that amplified bands from diverse materials were clear, stable, and rich in polymorphisms, indicating that the optimized system was very stable.

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