一个RNAi载体上反向重复片段的测序策略

相邻的反向重复DNA片段有形成单链内二级结构的倾向,属于一种测序困难的DNA模板。解决RNAi载体插入的反向重复片段的测序问题,为该类载体正确性的测序验证奠定基础。采用常规分子克隆方法构建表达小麦TaATG2串联反向重复片段的RNAi载体,设计2种策略对经菌落PCR初步鉴定的载体进行测序验证:一种是以完整的载体质粒为模板进行测序;另一种是先对载体进行酶切处理,切除反向重复片段中的一个后对保留另一个片段的线性载体进行测序。结果表明,第一种测序策略受到串联反向重复片段形成的单链内部二级结构的影响,测序信号在反向重复片段处出现衰减或乱峰,无法读取序列。第二种测序策略排除了2个反向重复片段之间的干扰,保留在载体上的片段测序信号清晰,序列准确。采用酶切切除一个片段后进行测序的方法,经过2次酶切和2次测序可以有效地对载体上的2个反向重复片段分别进行序列测定,进而确认构建载体的正确性。

A Sequencing Strategy for Inverted Repeats in RNAi Vectors

Adjacent inverted repeats in DNA tend to form secondary structure in a single strand,which are difficult templates for sequencing.The objective of this work is to solve the difficulty in sequencing inverted repeats inserted in RNAi vectors,and thus to lay a foundation for the verification of such vectors through DNA sequencing.A routine method of molecular cloning was adopted to construct an RNAi vector expressing tandem reverse repetitive fragments of wheat TaATG2 gene.Colony PCR was used to preliminarily identify the constructed vector,and 2 sequencing strategies were designed in this study:One was to use intact plasmids as sequencing templates,and the other was to use linearized plasmids as sequencing templates in which one of the reverse repetitive fragments was removed by digestion.The results showed that the sequencing reaction of the first strategy was influenced by secondary DNA structures formed within the region of reverse repetitive fragments.The sequencing signals from the reverse repetitive fragments were very weak or displayed chaotic peaks.The second strategy eliminated the interference between the two reverse repetitive fragments.Under this strategy,clear sequencing signals and accurate sequences were obtained for each fragment retained on the vector.The two reverse repetitive fragments inserted in an RNAi vector can be separately and effectively sequenced by 2 times of digesting and sequencing under a strategy of sequencing one fragment in the vector after removing the other by digestion.