马蔺ISSR-PCR反应体系的建立与优化

Optimization of ISSR-PCR System of Iris lactea by Uniform Design

以内蒙古呼和浩特市近郊野生马蔺DNA为模板,采用均匀设计,通过5因素5水平和5因素3水平两轮均匀优化试验,对影响ISSR-PCR扩增结果的一些因素(Mg2+、dNTP、引物、TaqDNA聚合酶和模板DNA浓度等)进行优化筛选,建立了适合马蔺的最佳ISSR-PCR反映体系:在25μl反应体系中,包括2.5μl 10×PCR Buffer、2.0mmol/L Mg2+、2.5μmol/L dNTP、1μmol/L引物1、.5U TaqDNA聚合酶、50ng模板DNA。在此基础上对扩增程序中的循环次数、退火温度进行筛选,获得的扩增程序为:94℃预变性3min;然后进行40个循环(94℃变性45s,52.3～60.5℃退火30s,72℃延伸1.5 min);循环结束后,72℃延伸5min。

Uniform design were amplified to optimize the influencing factors of ISSR-PCR system,including Mg^2＋,dNTP,primer,TaqDNA polymerase and DNA template concentration.The suitable ISSR-PCR system and ISSR-PCR procedure for Iris lactea of Hohhot were established as follows：each 25μl ISSR-PCR amplification reaction solution was consisted of 2.5μl PCR Buffer,2.0mmol/L Mg^2＋,2.5μmol/L dNTP,1μmol/L primer,1.5U TaqDNA polymerase and 50ng template DNA.The suitable ISSR-PCR procedure was 1 cycle of denaturing for 3 min at 94℃,40 cycles of denaturing for 45s at 94℃,annealing for 30s at 52.3～60.5℃,extending for 1.5 min at 72℃ and the last cycle of reextending for 5 min at 72℃.