摘要
[目的]分析影响木豆RAPD-PCR反应中的主要因素,优化反应条件。[方法]以木豆品种ICPL87091为试材,以木豆基因组DNA为模板,通过对PCR反应体系中各种参数的优化设置,分析比较各种因素对RAPD扩增结果的影响,建立适宜的反应体系。[结果]试验得到了较为理想的适宜木豆的反应体系。优化的木豆RAPD反应条件为:模板DNA浓度30 ng,随机引物1.6μmol/L,dNTPs(dATP,dCTP,dGTP,dTTP)各0.2 mmol/L,Mg2+浓度2.0 mmol/L,Taq酶1.0 U,反应体积为25μl。循环体系为:先94℃1 min,35℃2 min,72℃2 min,5个循环;然后94℃30 s,37℃1 min,72℃1 min,35个循环;最后72℃延伸10 min。[结论]利用这一反应体系可有效地进行木豆随机扩增多态性DNA分析,极大地提高了实验结果的可重复性。
[Objective] The aim was to analyze main factors affecting the RAPD-PCR reaction of pigeonpea and optimize reaction condition.[Method] With pigeonpea variety ICPL87091 as the tested material and the DNA of pigeonpea genome as the template,the various parameters in RAPD reaction system were optimized in design to compare the effect of each factor on RAPD amplification results and establish the suitable reaction system.[Result] An optimal reaction system suitable for the assay and usage of RAPD in pigeonpea was established.The final optimized pigeonpea RAPD reaction conditions was as follows： 30 ng template DNA,1.6 μmol/L random primer,0.2 mmol/L of each dNTP including dATP,dCTP,dGTP,dTTP,2.0 mmol/L Mg2＋ and 1 unit Taq DNA polymerase in a volume of 25 μl.The cycle system was devised as follows： 5 cycles of 94 ℃ for 1 min,35 ℃ for 2 min,72 ℃ for 2 min;35 cycles of 94 ℃ for 30 s,37 ℃ for 1 min,72 ℃ for 1 min;at last extension at 72 ℃ for 10 min.[Conclusion] Using this Reaction system could effectively
make the analysis of randomly amplified polymorphic DNA in pigeonpea and greatly increase the reproducibility of the test result.
出处
《安徽农业科学》
CAS
北大核心
2008年第20期8489-8491,共3页
Journal of Anhui Agricultural Sciences
基金
广西农科院院科技发展基金(2002003)资助
