马铃薯RAPD标记的PCR技术影响因素的探讨

FACTORS IN PCR REACTION SYSTEM INFLUENCING POTATO RAPD MARKERS

试验采用不同的马铃薯材料进行ＰＣＲ 技术体系的研究， 建立和完善了马铃薯少量材料的ＤＮＡ提取方法， 研究了ＰＣＲ扩增中Ｍｇ２ ＋ 浓度、引物和底物浓度对ＰＣＲ产物的影响。试验结果表明， 在以引物为１０ｍｅｒ 的ＰＣＲ反应体系中， 适当提高Ｍｇ２ ＋ 浓度（１－５ ～２－７ ｍＭ） 有利于ＰＣＲ反应的进行， 而引物与底物只有在一个相对适当浓度范围内才能获得理想的结果， 过低会使扩增产物达不到可检测的水平， 过高则会影响扩增产物的特异性。本试验中引物的适宜浓度为０－８ μＭ， 底物为８０ ～１８０ μＭ。所建立的优化马铃薯ＰＣＲ 体系同样适用于水稻、玉米、番茄和油菜等作物。

Different potato cultivars were involved in the experiment aiming at to establish a stable and efficient PCR system for potato RAPD markers.An improved protocol of DNA extraction with small amount of potato plant materials was developed.The effects of concentrations of Mg 2+ ,primer and substrate (dNTP)were investigated.The results showed that,in a PCR system with 10 mer primers,a relatively high concentration of Mg 2+ (1 5～2 7 mM in the experiment)insured a good result of the PCR reaction while a proper concentration of the primer and substrate should be obtained,which were 0 8 and 80～180 μM respectively,lower produced undetectable bands and higher affected the specificity of amplified products.The obtained optimized PCR system was approved with DNA samples of the other crops such as rice,maize,tomato and oil seed rape.