香加皮ISSR-PCR条件体系的优化

Optimization of ISSR-PCR System of Cortex Periplocae

采用正交设计法、均匀设计法,对影响香加皮ISSR-PCR体系的引物、Mg2+、dNTP、TaqDNA聚合酶浓度进行优化,通过两种方法的比较建立了适合香加皮ISSR-PCR的反应体系:在20μL反应体系中,含Mg2+1mmol/L、dNTP0.15mmol/L、引物1.0μmol/L、TaqDNA聚合酶1U、buffer2.5μL和模板DNA约50ng。在此基础上对扩增程序中的退火温度进行筛选,通过比较将退火温度定为50℃,最终扩增程序定为:94℃预变性4min接着进行40个循环:94℃变性30S,50℃退火1min,72℃延伸90S;循环结束后72℃延伸10min。

Orthogonal design and Uniform design was applied to optimize ISSR（inter-simple sequence repeat）-PCR（polymerse chain reaction ） system of Cortex Periplocae.Four influencing factors on ISSR- PCR,including primer,Mg2＋,dNTP,and Taq DNA polymerse consentration was tested.A suitable ISSR-PCR system for Cortex Periplocae was established as follows that each 20μ L ISSR-PCR amplification reaction solution was consisted of lmmol/LMg2＋, 0.15 mmol/L dNTP, 1.5μmol/L primer, lU Taq DNA polymerse and about 50 ng template DNA .The suitable ISSR-PCR Procedure was 1 cycle of denaturing for 4 min at 94℃ ,40 cycle of denaturing for 30S at 94℃ ,annealing for 1 min at 50℃ ,extenging for 90S at 72℃ and the last cycle of extenging for 10 min at 72℃ .