摘要
结合均匀试验与正交试验对影响目标区域扩增多态性—聚合酶链式反应(TRAP-PCR)效果的5个关键因素(DNA模板、Mg2+、dNTPs、引物及Taq酶)进行优化研究,建立了适用于凹叶厚朴目标区域扩增多态性(TRAP)分析体系。凹叶厚朴TRAP-PCR最优反应体系:总体积25μL,含模板DNA 40 ng,Mg2+1.5 mmol·L-1,dNTPs 0.28 mmol·L-1,固定引物及随机引物0.3μmol·L-1,Taq DNA聚合酶0.75 U。PCR扩增程序:94℃预变性5 min;94℃变性1 min,40℃复性1 min,72℃延伸1 min,5个循环;将退火温度升至50℃,其它条件不变进行38个循环;72℃延伸10 min。利用所得优化体系对16个不同种源凹叶厚朴进行扩增,结果表明该体系效果较好,可应用于凹叶厚朴相关研究。
Uniform design and orthogonal design methods were applied in this paper to optimize 5 key factors （ template DNA, Mg2＋, dNTPs, primers and Taq polymerase） that could influence the effect of TRAP-PCR.The TRAP-PCR system was set up in Magnolia officinalis subsp.biloba.The optimal system is in 25 μL reaction volume containing about 40 ng template DNA , 1.5 mmol· L^-1 of Mg2＋, 0.28 mmol· L^-1 of dNTPs, 0.3 μmol· L^-1 of fixed primer and arbitrary primer , and 0.75 U Taq DNA polymerase.PCR program：predenaturing 5 min at 94 ℃; the first five cycles run at 94 ℃, 1 min, 40 ℃, 1 min, and 72 ℃, 1 min, for denaturing , annealing and extension , respectively; then the annealing temperature is raised to 50 ℃ for another 38 cycles;run 10 min at 72 ℃for extension .Using the optimization system for amplification of 16 different provenances of M.officinalis subsp.biloba, results showed that the system had good effect and could be used in the correlational studies of M.officinalis subsp.biloba.
出处
《福建林学院学报》
CSCD
北大核心
2014年第2期165-170,共6页
Journal of Fujian College of Forestry
基金
福建省科技重点基金项目(2009N003)
国家"十二五"科技支撑项目(2011BA101B06)
关键词
凹叶厚朴
均匀试验
正交试验
优化
TRAP
Magnolia officinalis susbsp.biloba
TRAP
uniform design
orthogonal design
optimization
