一种简易的甘蔗叶组织PCR模板制备方法

A Simple Method for Preparing PCR Template of Sugarcane Leaf Tissue

以转基因甘蔗叶片为材料,少量嫩叶经碱并短暂高温处理,再中和,形成裂解混合物。直接以此为模板,转基因甘蔗外源基因bar、KP4、Cry1Ac-2A-gna融合基因及内源基因Shactin基因为靶基因,PCR扩增结果稳定、准确、重复性强,完全可以达到常规CTAB法提取的DNA扩增效果。用此方法制备的模板室温下2周之内,4℃、-20℃下一个月之内结果不变。经多种外源基因PCR反应的反复验证,证实了这种方法在转基因甘蔗检测中的广泛适用性。该方法快速制备PCR模板,无需DNA提取过程,具有使用样品量少,成本低、简便、快捷等优点。

Using transgenic sugarcane leaves as materials,a small amount of young leaves were treated by alkali and transient heat,then neutralized,and cracking mixture was formed. The mixture was directly used as the template of PCR,and transgenic sugarcane exogenous gene of bar,KP4,Cry IAc-2A-gna and endogenous gene Shactin as target genes,the amplified results were stable,accurate and reproducible,which reached the same effect of DNA amplification by conventional CTAB method. The templates by this method were still stable at room temperature for 2 weeks and at 4℃,-20℃ for 1 month. A series of exogenous gene PCR reactions were tested,which verified that this method was widely available in the detection of transgenic sugarcane. The method is fast for preparation of PCR templates without DNA extraction process,owing the advantages of requiring less material sample,low cost,simple,rapid,and efficient.