藏系绵羊随机扩增多态性DNA最佳反应体系的研究

Study on the best conditions of random amplified polymorphic DNA in Tibet sheep

以高原型藏羊的基因组DNA为模板,通过对RAPD反应体系中的各种参数进行优化试验,建立了适宜于藏系绵羊RAPD分析的最佳反应体系:在PE2400型DNA扩增仪的25μL反应体系中,模板DNA的量为60～120 ng,引物量为4～8pmol,Mg2+浓度为2.0 mM,Taq DNA聚合酶为1～2.5 U,dNTP浓度为150～200μM;94℃预变性3分钟后35次循环的参数设定为:94℃1分钟,36℃1分钟,72℃2.5分钟,最后72℃延伸10分钟。利用这些条件对藏系绵羊的基因组DNA进行RAPD分析,其结果的重复性和可靠性大为提高。

Based on the genomic DNA extracted from Tibet sheep, some essential factors were tested and optimized. It was established that the best reaction system was suitable for the analysis of RAPD in Tibet sheep. For a 25 μL reaction system in PE2400 DNA Amplifier, the optimal condition included 60-120 ng template DNA,4- 8 pmol arbitary primer, 2.0 mM Mg2+ , 1 - 2.5 U Taq DNA polymerase and 150 - 200 μM dNTP. The optimal program for efficient amplification included 35 cycles of denaturation Imin at 94 ℃ (3 min at 94 ℃ for the first cycle), 1min at 36℃ , 2min and 30sec at 72 ℃ , and 10min at 72 ℃ in the final extension. The optimal condition with good reproducibility and high reliability could be effectively used for RAPD analysis of Tibet sheep.