摘要
目的建立并优化青天葵样品总DNA提取方法及随机扩增多态性DNA(RAPD)反应。方法采用改进的高盐低pH法提取青天葵鲜叶和药材样品,通过正交设计和均匀设计,改变反应体系和扩增程序优化随机扩增多态性DNA。结果使用高盐低pH值法可较好地提取新鲜叶片的DNA,药材叶片含杂质较多,但都能用于RAPD。反应体系为:缓冲液2.0μ1,0.5μl dNTP(各2.5 mmol.L-1),0.8μlMg2+(25 mmol.L-1),0.5μl引物(20 pmol.μl-1),0.2μlTaq酶(5U.μ1-1),1μl DNA(50 ng),1μl BSA(20 mg.ml-1),加双倍蒸馏水至20μl。扩增程序为:94℃预变性3 min;94℃变性30 s,40℃退火30 s,72℃延伸60 s,40个循环;72℃延伸5 min。结论建立并优化了的青天葵样品总DNA提取方法及RAPD反应体系。
Objective To establish and optimize the methods of total DNA extraction and RAPD analysis of Nervilia fordii. Methods Low pH extraction medium with high salt method was adopted to extract genomic DNA from fresh and medicinal material leaves of Nervilia fordii, and the randomly amplified polymorphic DNAs （RAPD）technique was optimized through changing PCR reaction system. Results Low pH extraction medium with high salt was better to extract fresh leaves than medicinal material leaves, but both of them could be suitable to RAPD. The optimal RAPD conditions was as follows： 20μl solution contains 1 × Buffer solution, 0.5μl dNTP（each2.5mM） ,0.8μl Mg^2＋ （25mM）,0.5μl Primer（20pmol·μl^-1 ）, 0.2μl Taq DNA polymerase （5U·μl^-1 ）, 1μl DNA （50ng） , 1μl BSA （20mg·ml^-1 ）. RAPD program was 3 min at 94℃ for predenaturation, then 40 cycles of 30 sec at 94℃ for denaturation, 30 sec at 40℃ for annealing, 1 rain at 72℃ for extension, finally extension at 72℃for 5 min. Conclusion The extraction methods for total DNA and RAPD analysis of Nervilia fordii have been established.
出处
《时珍国医国药》
CAS
CSCD
北大核心
2009年第6期1431-1432,共2页
Lishizhen Medicine and Materia Medica Research
基金
广东省自然科学基金项目(No.5004249)
广东省科技计划项目(No.2004B330001009)
