台湾水青冈ISSR-PCR体系的优化

分析了ISSR-PCR体系中Mg2+、dNTP和BSA浓度,以及模板DNA、引物和TaqDNA聚合酶用量等6个因素对反应结果的影响,建立了适合于台湾水青冈ISSR-PCR扩增的反应体系:10μL PCR反应液中,含10 ng DNA,1.5 mmol.L-1Mg2+,0.125 mmol.L-14×dNTP,1×TaqDNA聚合酶配套缓冲液(10 mmol.L-1Tris-HCl,pH值9.0,50 mmol.L-1KCl,0.1%Triton X-100),0.6 UTaqDNA聚合酶,2 mg.mL-1BSA和3pmol引物。应用优化的ISSR-PCR体系从100个ISSR引物中筛选出12个稳定性好,重复性高的引物,对22株台湾水青冈个体的DNA进行扩增,结果表明优化的ISSR-PCR体系完全适合于台湾水青冈的ISSR分析。

Optimization of ISSR-PCR System of Fagus hayatae

This paper analyzed the influence of the 6 factors on the reaction results,the factors included:the concentration of Mg2+,dNTP and BSA in ISSR-PCR system,as well as the dosage of the template DNA,primer and Taq DNA polymerase.It established the response system suitable for ISSR-PCR amplification of Fagus hayatae:10μL of PCR reaction solution,including 10 ng of DNA,1.5 mmol·L-1 of Mg2+,0.125 mmol·L-1 of 4 × dNTP,1×Taq DNA polymerase matching buffer(10 mmol·L-1 Tris-HCl,pH value of 9.0,50 mmol·L-1 of KCl,0.1% of Triton X-100),0.6 U of Taq DNA polymerase,2 mg·mL-1 of BSA and 3 pmol primer.With the application of optimized ISSR-PCR system,12 stability,high repeatability of primers were selected from the 100 ISSR primers.DNA amplification was carried out on 22 F.hayatae individuals,the results showed that the optimization of ISSR-PCR system was completely suitable for F.hayatae ISSR analysis.