利用正交设计优化水曲柳ISSR-PCR反应体系

Optimization for ISSR-PCR System of Fraxinus mandshurica Rupr.Using Orthogonal Design

利用正交设计L16(45)对水曲柳ISSR-PCR反应体系的5因素(Taq酶,Mg2+,模板DNA,dNTP,引物)在4个水平上进行优化试验,PCR结果用统计软件MINITAB分析:①各因素的不同水平对PCR反应结果都有显著的影响,其中Taq酶量影响最大;②筛选出各反应因素的最佳水平,建立水曲柳ISSR-PCR反应的最佳体系(20滋l)为:Taq酶1.0U,Mg2+2.0mmol/L,模板DNA50￣200ng,dNTP0.15mmol/L,引物0.3滋mol/L。最后,对水曲柳ISSR-PCR最佳反应体系进行梯度退火,得到最佳退火温度为58.3℃。这一优化系统的建立为今后利用ISSR标记技术,研究水曲柳的地理变异提供一个标准化程序。

In this paper, the orthogonal design was used to optimize ISSR-PCR ampl ification system on Fraxinus mandshurica Rupr. in four levels of five factors (T aq DNA polymerase, Mg2+, DNA template, dNTP, and primer) respectively. The resul t of PCR was analyzed by software MINITAB, and the result showed that: ①The aff ections of each factor in different levels were found and the quantity of Taq DN A polymerase was the most effective factor to the result of PCR; ② A most suita ble ISSR-PCR system for Fraxinus mandshurica Rupr. was established, it's 20?滋l reaction system which contained 1.0U Taq DNA polymerase, 2.0mmol/L Mg2+, 50~200n g DNA template, 0.15mmol/L dNTP, and 0.3?滋mol/L Primer. The optimal annealing t emperature for ISSR-PCR reaction was proposed by gradient PCR and it was 58.3℃. The result provided a standardizing ISSR-PCR program for the analysis of geogra phic variation of Fraxinus mandshurica Rupr.