红花SCoT-PCR反应体系的建立与优化

Establishment and Optimization of SCoT-PCR Amplification System in Safflower

为构建红花最佳扩增体系,为以后研究红花遗传多样性和目的基因标记提供了科学依据。本研究采用L_(16)(4^(5))正交试验和单因素试验相结合的方法对红花SCoT-PCR反应体系中的引物,DNA、dNTPs、PCR Buffer (含Mg^(2+))、Taq DNA聚合酶进行优化,每个因素都设为四水平。结果表明,五个影响因素中dNTPs的影响最大,红花最佳反应体系为引物0.6 mmol/L,DNA 60 ng,dNTPs 0.25 mmol/L,Taq DNA聚合酶1.5 U,PCR Buffer (含Mg^(2+)) 2μL,ddH_(2)O补足至20μL。选用引物S15对八种红花资源对扩增体系进行验证,结果显示扩增条带清晰,多态性好,表明该体系适合红花分子标记研究。

To construct the safflower optimal amplification system,and lay a foundation for the future study on the genetic diversity and target gene marker of safflower.We applied the combination of L_(16)(4^(5)) orthogonal experiment and single factor experiment to optimize the primer,DNA,dNTPs,PCR Buffer (containing Mg^(2+)),Taq DNA polymerase in the SCoT-PCR reaction system of safflower.Each factor was set at four levels.The results showed that d NTPs had the greatest effect among the five factors,and the optimal reaction system of safflower was primer 0.6 mmol/L,DNA 60 ng,dNTPs 0.25 mmol/L,Taq DNA polymerase 1.5 U,PCR Buffer(containing Mg^(2+)) 2μL,and ddH_(2)O supplemented to 20μL.Using the primer S15,selection of 8 safflower resources were established to verify the amplification system,which showed that the amplified bands were clear and the polymorphism was good.The results showed that the system was suitable for the molecular marker study of safflower.