正交优化法建立沙打旺ISSR-PCR最佳反应体系

根据正交试验设计的原理,设计了探索性正交试验和细调性正交试验来确定沙打旺ISSR-PCR体系中各成分的浓度。得到既稳定又能扩出最多条带的适合沙打旺的ISSR-PCR最佳反应体系,即20μl的反应体系中含有1×buffer,dNTP 0.2 mmol/L,Taq酶1.0 U,引物0.3μmol/L,Mg2+2.5 mmol/L,DNA模板2.5 ng/μl。然后对沙打旺ISSR-PCR最佳反应体系进行梯度退火试验,得到这条引物的最佳退火温度为54.1℃。这一最佳体系的建立为今后利用ISSR标记技术,研究沙打旺的遗传多样性提供了标准化程序。

Establishment of Astragalus adsurgens ISSR-PCR Optimal Conditions with Orthogonal Optimization Method

Orthogonal design is applied to the ISSR-PCR conditions optimization in this paper.Two orthogonal experiments were made to investigate the amplification efficiency of different amplification conditions.As a result,a satisfactory ISSR technique system for Astragalus adsurgens with desirable repeatability and polymorphic bands was established.In a total volume of 20?μl ISSR-PCR system,it contained 1×buffer,0.2?mmol/L dNTP,0.3?μmol/L Primer,2.5?mmol/L Mg~(2+),1U Taq DNA polymerase and 2.5?ng/μl template DNA.The optimal annealing temperature of this primer for ISSR-1PCR reaction was proposed by gradient PCR and it was 54.1?℃.The result provided standardizing ISSR-PCR program for the analysis of genetic diversity of Astragalus adsurgens.