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Sanger测序法确证硫代修饰反义脱氧寡核苷酸的序列 被引量：1

Sequence confirmation of phosphorothioate antisense oligodeoxynucleot ides using direct Sanger sequencing reactions
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摘要 目的 :建立一种简便、可靠的硫代修饰反义脱氧寡核苷酸序列确证方法 ,为反义药物的质量控制及新药研究与开发奠定基础。方法 :选择含有待测反义脱氧寡核苷酸序列在内的一段靶基因为模板 ,以待测序的硫代修饰反义脱氧寡核苷酸作为下游引物 ,同时在其上游设计一条引物 ,进行PCR扩增 ,使待测序的反义序列以修饰形式掺入到PCR扩增产物中 ;以上述掺入反义序列的PCR扩增产物为模板 ,利用Sanger双脱氧链终止测序法对其进行自动序列分析。结果 :该方法可对不同长度硫代修饰的反义序列进行测定 ,并且可有效地检测出合成过程中产生的突变和缺失序列 ,与质谱法相比 ,仪器和试剂更普及 ,方法易于掌握且测序成本低 ,用样量少。结论 :该方法可用于硫代修饰反义脱氧寡核苷酸药物的序列确证。 Objective: To establish a method for conve ni ent and reliable sequence confirmation of phosphorothioate antisense oligodeoxyn ucleotides. Methods: The gene region containing the antise nse oligodeoxynucleotide (ASODN) was selected as the template. The target region was PCR amplified using phosphorothioate ASODN and a designed forward primer.Th e direct DNA sequencing of PCR product containing the phosphorothioate modified antisense sequence, was employed based on the Sanger dideoxynucleotide chain-t e rminating DNA sequencing reaction. Results:Direct sequenci ng of the modified phosphorothioate backbone of oligodeoxynucleotides was not on ly applicable to measurement of different length of ASODN, but also could detec t mutation, deletion and insertion during synthesis process. Compared with MS se q uencing, this method was more convenient in operation and lower in cost and nee d ed fewer amount of sample. Conclusion:The direct sequencin g method is an easy method for routine sequence analysis in antisense drug quali ty control.
作者 陈苏红 鲁丹丹 张嵬 王升启
机构地区 军事医学科学院放射医学研究所
出处 《军事医学科学院院刊》 CSCD 北大核心 2004年第5期442-445,497,共5页 Bulletin of the Academy of Military Medical Sciences
基金 国家"8 63"计划基金 (2 0 0 2AA2Z3 3 3 7) 国家自然科学基金 (3 0 17111)资助
关键词 硫代反义脱氧寡核苷酸 序列分析 反义 寡核苷酸 phosphorothioate antisense oligodeoxynucleo tide sequence analysis antisense oligodeoxynucleotide
分类号 R346 [医药卫生—基础医学]
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参考文献14

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军事医学科学院院刊
2004年 第5期

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