Sanger测序法确证硫代修饰反义脱氧寡核苷酸的序列

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。

Sequence confirmation of phosphorothioate antisense oligodeoxynucleotides using direct Sanger sequencing reactions

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.