石鲽基因组DNA的提取及其RAPD体系的优化  

DNA Extraction from Kareius bicoloratus Genome and Its Optimization of RAPD Reaction System

作　　者：徐建鹏[1] 赵彦宏[1] 赵建萍[1] 刘林德[1] 孙振兴[1] 万军利[1] 王爱云[1] 常林瑞[1] 李清[1] 

机构地区：[1]鲁东大学生命科学学院,山东烟台264025

出　　处：《安徽农业科学》2008年第35期15377-15378,15583,共3页Journal of Anhui Agricultural Sciences

基　　金：鲁东大学校人才基金(LY20083304)资助

摘　　要：[目的]研究石鲽基因组DNA的提取及其RAPD体系的建立和优化。[方法]以石鲽为试材,按常规酚/氯仿抽提法提取其基因组的DNA,对影响RAPD反应的各因素进行优化,建立了石鲽的最佳RAPD反应体系和程序。[结果]采用常规酚/氯仿抽提法获得的DNA完全能够满足RAPD分析的要求。通过优化建立一套适合石鲽的稳定的RAPD反应体系:反应体系总体积为25μl,包括10×buff-er2.5μl,MgCl22 mmol/L,dNTPs 0.15 mmol/L,引物0.2μmol/L,模板30 ng,Taq酶1 U。扩增程序为:94℃预变性5 min,45次PCR循环(94℃变性45 s,36℃退火45 s,72℃延伸2 min和72℃延伸10 min。利用该体系对OPK和OPV系列共40条引物进行扩增,发现其中部分引物能产生稳定、清晰的条带。[结论]该体系为石鲽遗传多样性以及相关分子标记的研究奠定了基础。[ Objective ] The study aimed to DNA extraction from Kareius bicoloratus genome and the establishment and optimization of RAPD reaction system. [ Method] The genomic DNA was extracted from K. bicoloratus individual according to conventional extraction method of phenol/chloroform. Based on the optimization of RAPD reaction conditions, the RAPD reaction system for K. bicoloratus was established. [ Result] DNA obtained by using the conventional extraction method of phenol/chloroform could fully meet the demand of RAPD analysis. A RAPD reaction system for K. bicoloratus was established through optimization, which was following ： the total volume of RAPD reaction was 25 bd, including 2.5 μl 10 × buffer, 2 mmol/L MgCl2 ,0. 15 mmol/L dNTPs ,0.2 μmol/L primer,30 ng template DNA and 1 U Taq DNA polymerase. The PCR program was as follows ： 5 rain pre - amplification denaturation at 94℃, 45 cycles of 45 s at 94 ℃ for denaturation, 45 s at 36 ℃ for annealing, 2 min at 72 ℃ for extension, finally extension at 72 ℃ for 10min. When this system was used to amplify 40 primers from OPK and OPV series, it was found that their part primers could produce the stable and clear bands. [ Conclusion] This system laid the foundation for further study of the genetic diversity and related molecular markers in K. bicoloratus.

关 键 词：石鲽 DNA提取 RAPD 优化 

分 类 号：S188[农业科学—农业基础科学]