摘要
以玉米开放群体为试材，对引物种类，复性温度，引物、ｄＮＴＰＳ、Ｍｇ＋＋、缓冲液、Ｔａｑ聚合酶浓度等因素对ＲＡＰＤ带型的影响进行了比较研究。结果表明，在不同复性温度下，有的引物在个体间扩增相同的带型，有的引物在个体间扩增不同的带型。同一引物高浓度比低浓度扩增信号强。高浓度ｄＮＴＰＳ比低浓度ｄＮＴＰＳ扩增效果差。低浓度Ｍｇ＋＋无扩增产物。高浓度Ｍｇ＋＋和高浓度聚合酶引起带纹模糊拖尾。高浓度缓冲液无扩增信号。双引物扩增可增加带数。根据以上研究结果，建立了适合玉米ＲＡＰＤ分析的技术体系：２５μｌ反应液中，含１倍缓冲液，１．５ｍｍｏｌ／ＬＭｇＣｌ２，０．２ｍｍｏｌ／ＬｄＡＴＰ，０．２ｍｍｏｌ／ＬｄＴＴＰ，０．２ｍｍｏｌ／ＬｄＣＴＰ，０．２ｍｍｏｌ／ＬｄＧＴＰ，０．８μｍｏｌ／Ｌ随机引物，１．５ＵＴａｑ酶，６０ｎｇ模板ＤＮＡ。
By using open-pollinated maize population as experimenting materials, a comparative study was made and results showed that, primer kinds, annealing temperatures and concentrations of primer, dNTPS, Mg￣(++), buffer and Taq polymerase were all factors affecting RAPD pattern. At different annealing temperatures, some primers amplified the same patterns among individuals, others amplified different patterns. For a same primer, higher concentration amplified stronger signals. High concentration of dNTPS had a weaker amplifying effect as against low concentration. No fragments were produced with low concentration of Mg￣(++) or high concentration of buffer. High concentration of Mg￣(++) and Taq polymerase resulted in bands smear. Paired primers could amplify new RAPD bands and detect more polymorphisms. Based on the above research,a technical system for RAPD analysis was established: in 25 μl reaction solution, contain 1×buffer,1.5 m mol/L MgCl2,0.2 m mol/L dATP,0.2 m mol/L dTTP,0.2m mol/L dCTP,0.2 dGTP,0.8m mol/L random primer,1.5UTaq polymerase, and 60 ng template DNA.
出处
《华中农业大学学报》
CAS
CSCD
北大核心
1996年第5期405-409,共5页
Journal of Huazhong Agricultural University
基金
国家自然科学基金
关键词
玉米
RAPD
影响因素
Zea mays, RAPD, affecting factors
