Optimization for ISSR-PCR reaction system on Schisandra henryi by orthogonal design / 中草药

ABSTRACT

Objective: To optimize the each factor affecting on ISSR-PCR reaction system of Schisandra henryi and establish the stable ISSR-PCR system. Methods: Based on the analysis of orthogonal design test, an orthogonal design was used to optimize the ISSR-PCR amplification system on S. henryi by five factors (Taq polymerase, Mg2+, DNA template, dNTP, and primer) at four concentration levels, respectively. Results: A suitable ISSR-PCR reaction system was constructed with the 20 μL reaction system containing 1.00 U Taq polymerase, Mg2+1.50 mmol/L, DNA template 40.00 ng, dNTP 0.25 mmol/L, and primer 0.50 μmol/L. Twelve effective ISSR primers were selected and the optional annealing temperature of every one primers was fixed. Conclusion: ISSR-PCR is significantly influenced by the concentration of S. henryi. This ISSR-PCR system could provide clear bands, reliable reaction system, and abundant polymorphisms. It proves a reference for molecular research of S. henryi.