NaCl浓度和茎环内的碱基结构对shRNA退火影响的研究

Effect of stem-loop base inside structures and NaCl concentration on shRNA annealing

目的优化DNA寡核苷酸单链退火条件,提高单链DNA寡核苷酸退火效率,加速shRNA表达载体构建速度。方法将茎环内部碱基结构不同DNA寡核苷酸链在不同浓度(50、100、150、200mmol/L)NaCl退火缓冲液中进行退火,通过琼脂糖凝胶电泳观察DNA寡核苷酸链在退火前后电泳速度的变化,利用Gene Tools分析软件对电泳图像进行分析比较退火效率。结果当退火缓冲液中NaCl浓度为200mmol/L时,DNA寡核苷酸单链退火效率明显提高,茎环内部碱基互补的DNA寡核苷酸单链退火效率46.00%±3.33%,茎环内部碱基不互补DNA寡核苷酸单链退火效率97.00%±1.49%。结论NaCl浓度和茎环内的碱基设计是影响shRNA退火主要因素,优化shRNA的退火条件,提高退火效率,可以加速载体构建。目的优化DNA寡核苷酸单链退火条件,提高单链DNA寡核苷酸退火效率,加速shRNA表达载体构建速度。方法将茎环内部碱基结构不同DNA寡核苷酸链在不同浓度(50、100、150、200mmol/L)NaCl退火缓冲液中进行退火,通过琼脂糖凝胶电泳观察DNA寡核苷酸链在退火前后电泳速度的变化,利用GeneTools分析软件对电泳图像进行分析比较退火效率。结果当退火缓冲液中NaCl浓度为200mmol/L时,DNA寡核苷酸单链退火效率明显提高,茎环内部碱基互补的DNA寡核苷酸单链退火效率46.00%±3.33%,茎环内部碱基不互补DNA寡核苷酸单链退火效率97.00%±1.49%。结论NaCl浓度和茎环内的碱基设计是影响shRNA退火主要因素,优化shRNA的退火条件,提高退火效率,可以加速载体构建。

Objective To optimize the annealing condition of single strand DNA oligonucleotide,and improve the annealing efficiency to accelerate the construction speed of shRNA expression vector.Methods The different single-stranded DNA oligonucleotides in stem-loop base inside structures were annealed in different NaCl concentrations（50,100,150,200 mmol/L）,the change of oligonucleotide electrophoresis speed in agarose gelelectrophoresis were observed,and its annealing efficiency were analyzed and compared with the GeneTools analysis software.Results The annealing efficiency of single-stranded DNA oligonucleotide was increased obviously when NaCl concentration in annealing buffer was 200 mmol/L,the annealing efficiency inside of stem-loop base complementary structure in single-stranded DNA oligonucleotide were 46.00 % ± 3.33 %,the inside base non-complementary structure of stem-loop annealing efficiency in single-stranded DNA oligonucleotide were 97.00 % ± 1.49 %.Conclusion The NaCl concentration and stem-loop design of the base are major factors in shRNA annealing.The shRNA optimized annealing conditions to improve the efficiency of the annealing can speed up the construction of shRNA expression vector.