萝卜基因组DNA的RAMP-PCR体系优化

Optimization of RAMP-PCR reaction system in genomic DNA of radish(Raphanus sativus)

以萝卜为材料，对基因组DNA的RAMP分析体系中的Mg^2＋、dNTPs和引物浓度进行优化。分别设计3个浓度梯度：Mg^2＋为0．75、1．5、3．0mmol·L^-1；dNTPs为0．05、0．15、0．3mmol·L^-1；引物为0．065、0．2、0．4μmol·L^-1，并对合适退火温度进行研究。筛选出的RAMP优化体系为（20μL）：dNTPs 0．15mmol·L^-1，Mg^2＋ 1．5mmol·L^-1，引物0．2～0．4μmol·L^-1，DNA 10ng，Taq E 0．8U；PCR扩增程序为94℃ 3min，94℃ 1min，45℃ 1min，72℃ 1．5min，42个循环，72℃ 8min。运用此体系，进行引物组合筛选，并对7个萝卜品种的遗传多样性与品种鉴定进行RAMP标记分析。

Genomic DNA of Radish was analyzed by optimizing the concentration of Mg^2＋ , dNTPs and primer in Random Amplified Microsatellite Polymorphism （RAMP） system. Three different concentration gradients were set respectively, Mg^2＋ 0.75, 1.5,3.0 mmol ·L^-1; dNTPs 0.05, 0.15,0.3 mmol·L^-1 ; primer 0. 065,0.2,0.4 μmol ·L^-1, and the suitable annealing temperature was also screened. An optimization of RAMP reaction system for radish genomic DNA is（20 μL） ： dNTPs 0.15 mmol ·L^-1 ,Mg^2＋ 1.5 mmol ·L^-1 ,primer 0.2 -0.4 μ~mol·L^-1 ,DNA 10 ng,Taq E 0.8U. The amplification protocol was： 94℃ 3 min,42 cycles for 1 min at 94℃ ,1 min at 45℃, 1.5 min at 72℃, followed by a final extension of 8 min at 72℃. The suitable system was applied in screening the primer combinations, and the genetic diversity of seven radish varieties was analyzed in RAMP marker system.