Establishment and optimization of RAMP-PCR reaction system for DNA of Eupolyphaga sinensis / 中草药

ABSTRACT

Objective: To provide the basis for the genetic diversity research of Eupolyphaga sinensis by establishing and optimizing the random amplified microsatellite polymorphism RAMP-PCR reaction system and amplification procedure for genomic DNA. Methods: Phenol chloroform extraction of genomic DNA was performed on female E. sinensis. Based on RAMP Primer (ISSR807 + RAPD A5), the orthogonal test was adopted to optimize the RAMP-PCR amplification system on E. sinensis in five factors (Mg2+, dNTPs, primer, Taq DNA polymerase, and template DNA) at four levels, and the suitable anneal temperature of the primer was selected. Results: The optimal Primer (ISSR807+RAPD A5) for RAMP-PCR system (25 μL reaction volume) of E. sinensis was 10 × PCR buffer (2.5 μL), Mg2+ (1.0 mmol/L), dNTPs (2.0 mmol/L), primer (0.5 μmol/L), Taq DNA polymerase (1 U), and template DNA (1.5 ng/μL); The optimized anneal temperature was 46.8°C. Conclusion: The established and optimized RAMP-PCR reaction system is stable and repeatable, which could provide the basis for the analysis of germplasm resources and genetic diversity of E. sinensis.