小麦RAPD分析中温度循环参数的优化

Optimization of the Temperature Profile for Wheat RAPD Analysis

为了解决小麦材料 RAPD分析中的重复性差、多态性低等问题 ,对反应中的温度循环参数进行了优化研究。通过比较不同变性时间、复性温度和升温速度等的扩增效果 ,设计出了适于小麦 RAPD分析的 PCR程序 :94℃预变性 5 m in;开始的 5个循环为 94℃ 1min,38℃ 1m in,0 .3℃ / s升温 ,72℃ 2 min;后 40个循环为 94℃ 10 s,38℃ 1min,0 .3℃ / s升温 ,72℃ 2 min;最后 72℃ 5 m in。在后 40个循环中用 1～ 2 s的变性时间 ,则能够有目的地扩增 6 0 0 bp左右及以下片段。利用该程序 ,每条引物检测的位点数平均达 17.6个左右 ,高于一般报道的 1～ 10个。该程序在小麦遗传多样性研究。

Optimization of the temperature profile was carried out in PCR for enhancing the reproducibility and polymorphism in wheat RAPD analysis. The effects were compared of denaturation time, annealing temperature and ramp time. The following temperature profile was advanced suitable for wheat RAPD analysis : initially at 94℃ for 5 min, 5 cycles at 94℃ for 1 min, at 38℃ for 1 min and at 72℃ for 2 min with a ramp time of 0.3℃ /s from 38℃ to 72℃, followed by 40 cycles at 94℃ for 10 s, at 38℃ for 1 min and 72℃ for 2 min with the same ramp time as in previous 5 cycles, at last keep at 72℃ for 5 min. In the subsequent forty cycles denaturation can be performed for 1~2 s to amplify about 600 bp and shorter fragments. Using this profile, the average of the loci detected by each primer was about 17.6, which was more than those (1~10) reported in references. This profile is practically valuable in wheat RAPD analysis for genetic diversity research , identification of molecular markers and so on.