红花随机扩增多态性DNA反应体系的建立与优化  

Establishment and optimization of reaction system of random amplified polymorphic DNA in Carthamus tinctorius L.

作　　者：张阵阵[1] 郭美丽[1] 张军东[2] 陆倍倍[1] 

机构地区：[1]第二军医大学药学院生药学教研室,上海200433 [2]第二军医大学药学院药理学教研室,上海200433

出　　处：《药学服务与研究》2006年第1期10-13,共4页Pharmaceutical Care and Research

基　　金：国家自然科学基金资助项目(No.30271588)

摘　　要：目的:考察不同因素对红花随机扩增多态性DNA反应体系的影响,建立并优化反应体系。方法:提取红花基因组DNA,分别设计Taq酶浓度(三水平:0.02、0.04、0.06 U/μL)、dNTP浓度(三水平:0.10、0.20、0.30 mmol/L)和引物(primer)浓度(三水平:0.16、0.32、0.48 pmol/L)的三因素实验,进行PCR扩增。根据PCR产物的琼脂糖凝胶电泳图确定最优的Taq酶浓度、dNTP浓度和引物浓度。进一步设计Mg2+浓度(三水平:1.0、1.5、2.0 mmol/L)和模板浓度(四水平:1.00、1.25、1.50、1.75 mg/L)的两因素实验,进行PCR扩增,确定最佳Mg2+浓度和模板浓度。结果:三因素实验中,第17条组合泳道和两因素实验中第6条组合泳道扩增主带稳定、均匀、清晰,条带数目多。结论:确定红花随机扩增多态性DNA反应25μL反应体系中最佳组合为:Taq酶0.04 U/μL、dNTP 0.30 mmol/L、引物0.32 pmol/L、Mg2+1.5 mmol/L、模板浓度1.00 mg/L。Objective： To study the effects of different factors on random amplified polymorphic DNA reaction system in Cartharnus tinctorius L. , and establish and optimize the reaction system. Methods： Genome DNA of Carthamus tinctorius L. was isolated. A three factor-experiment of Taq enzyme concentrations （0.02,0.04,0.06 U/μL）, dNTP concentrations （0.10, 0.20, 0. 30 mmol/L） and primer concentrations （0. 16, 0.32, 0.48 pmol/L） for PCR amplification were designed. According to the result of agarose gel electrophoresis test,the optimal concentrations of Taq enzyme, dNTP and primer were determined. In addition, a two factor-experiment of Mg^2＋ concentrations （ 1.0,1.5,2.0 mrnol/L） and template concentrations （ 1.00,1.25, 1.50,1.75 mg/L） for PCR amplification were designed. According to the result of agarose gel electrophoresis test, the optimal concentrations of Mg^2＋ and template were determined. Results： The seventeenth band in the three factor-experiment and the sixth band in the two factor-experiment were stable, homogeneous and clear. Conclusion： The optimal reaction system of random amplified polymorphie DNA in Cartharnus tinctorius L. was determined as Taq enzyme 0.04 U/μL, dNTP 0.30 mmol/L, primer 0. 32 pmol/L, Mg^2＋ 1.5 mmol/L and template 1.00 mg/L.

关 键 词：红花 随机扩增多态性DNA反应体系 构建 

分 类 号：Q943[生物学—植物学]