作　　者： ; ; ; ; ; ; ; ; ;

机构地区： 南方医科大学公共卫生与热带医学学院病毒研究所

出　　处： 《热带医学杂志》 2010年第3期253-257,共5页

摘　　要： 目的通过对重叠延伸PCR技术的改进,建立一种可用于多DNA片段连接的SOE-PCR方法。方法扩增获得相互重叠的1.8、1.1、1.3kb的短片段,以之作为亚片段模板。在SOE-PCR反应体系中加入不同数量用于重叠的亚片段模板及延伸所得目的长片段扩增所需的外引物,同时加入了稀释100倍的内引物,使得亚片段模板在扩增过程中能动态达到最佳重叠延伸浓度。结果获得了特异性强、丰度高的两段连接产物,与原有的SOE-PCR方法相比,大大减小了反应的非特异性条带扩增,产物丰度也优于传统的扩增方法。采用同样方法尝试了单管反应对三个片段进行连接,获得了总长度为4.2kb的长片段,且实验重复性、特异性好。结论本研究以相互重叠的多个短片段为模板,实现多片段的连接和扩增,可降低受模板浓度条件的影响的实验操作难度。多片段连接时减少了操作步骤和突变几率,可广泛用于多DNA片段连接操作。 Objective To discuss the SOE-PCR technique in long piece or multiple pieces gene mutations operation.Methods After the primary PCR reaction,besides putting the flanking primers of recommended concentration,we changed the traditional way into putting 100-time diluted internal primers which used to amplify the primary PCR templates.Through such operations,we can achieve the most satisfied templates concentration of SOE-PCR dynamically.Results Compare with the traditional SOE-PCR,two pieces combination products with the new way is more stable and specific while the specific abundance ratio keeps well.At the same time we achieve the three pieces combination with a total length of 4.2 kb in the same reaction while the specific amplification abundance also satisfied.Conclusion With the new method,we can overlap pieces combination of SOE-PCR in the same reaction tube with the specificity and abundance keeps well while not like the traditional way of combine two pieces together.When in multiple chimerical genes operation,this kind of method can reduce PCR reaction cycles,thus may be meaningful for chimerical genes operations.

关 键 词： 重叠延伸 突变 多片段连接

领　　域： [生物学]