摘要
采用交互正交设计L64(421)对濒危植物云南红豆杉简并锚定微卫星-PCR反应体系分别进行了5因素(Taq酶;Mg2+;dNTP;DNA及引物)4水平的优化筛选,SAS软件统计分析表明:3个单因素(Taq酶;Mg2+;dNTP)和2个交互作用(Taq×Mg2+,Mg2+×dNTP)对此反应体系有显著影响作用。云南红豆杉简并锚定微卫星-PCR反应的优化体系是:在20μL反应体系中含有1×PCR buffer,Taq酶4 U,Mg2+(2.0 mmol.L-1),dNTP(0.4 mmol.L-1),DNA75 ng,引物(1.0μmol.L-1),退火温度(54℃)。交互正交设计既能快捷有效地筛选出最佳PCR反应体系,又能揭示试验因素及其交互作用对扩增的影响程度,分析结果更客观、准确,具有一定的应用价值。
The interaction among different factors was usually ignored during experimental design with orthogonal design which was widely used in optimizing for PCR reaction system. Orthogonal design L64 （4^21 ）involving interaction was used to optimize simple sequence repeated-anchored PCR by degenerate primer（ SSR- anchored PCR） amplification system on T. yunnanensis in five factors： Taq DNA polymerase, Mg^2＋ , dNTP, DNA and primer at four levels. The analytic results by software SAS showed that：①Three factors（ Taq DNA polymerase, Mg^2＋ , dNTP） and the two interactions （Taq× Mg^2＋ , Mg^＋2× dNTP） had notable effect on the SSR-anchored PCR amplification system ;② An optimal SSR-anchored PCR reaction system was established containing 1× PCR buffer,4 U Taq DNA polymerase, 2.0 mmol· L^-1Mg2^＋ , 0.4 mmol·L^-1dNTP,75 ng DNA,1.0 μmol · L^-1 primer in a total volume of 20 μL reaction system. Moreover the optimal annealing temperature（54 ℃） was proposed by gradient PCR. Orthogonal design involving interaction is of special application value as it can not only screen out the best PCR reaction system quickly and effectively, but also reveal the extent to which the testing factors and their interaction can affect the PCR amplification, and thus make the analytic result more objective and accurate. While, further research of the judgmental standard to the electrophoretogram of PCR amplification products is still needed to improve both the facility of the method and the reliability of experimental data.
出处
《林业科学研究》
CSCD
北大核心
2007年第6期739-743,共5页
Forest Research
基金
科技部科研院所公益研究专项项目"濒危植物云南红豆杉的资源及保护技术研究"(2004DIB3J104)
国家科技支撑项目"特种工业原料林培育技术"(2006BAD18B03)的部分研究内容
