【摘要】 为满足烟草遗传分析的需求,建立稳定的IMP标记扩增方法,对IMP分子标记体系进行了优化。以烟草DNA为模板,利用Li-cor4300凝胶分离系统,优化了模板、Mg2+、dNTP、聚合酶浓度等IRD荧光引物扩增反应体系主要参数,建立分析IMP标记的平台。结果显示,影响烟草荧光引物扩增反应最大的因素是dNTPs和引物浓度,反应体系对Mg2+浓度的要求较宽泛,同时,不同的引物对各因素的浓度要求也有差异。最终确定20μL反应体系中,模板DNA70ng,Mg2+浓度2.0mmol/L,dNTPs为0.2mmol/L,rTaqDNA聚合酶1.5U,引物浓度均为1μmol/L。该体系扩增条带稳定,重复性好,适合烟草遗传多样性分析。更多还原

【Abstract】 The amplification between two adjacent MITEs was called Inter-MITE polymorphism(IMP).To meet the demand of tobacco genetic analysis and establishment of stable IMP amplification method,the IMP PCR system was optimized using IRD primer.In this study,every major amplification parameter such as template,Mg 2 +,dNTP,rTaq concentration were optimized and analyzed by Li-cor 4300 system based on tobacco genome DNA.The results showed the optimized PCR-amplifications were performed in a 20 μL volume containing 70 ng template DNA,2.0 mmol/L of MgCl 2 +,0.2 mmol/L of dNTPs,1 μmol/L of labeled primer and 1.5 U of rTaq DNA polymerase.The results demonstrates that the optimized IMP method is stable robust and can be effectively applied in tobacco genetic diversity analysis.更多还原