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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 simple and reliable method for confirming the sequence of antisense oligodeoxynucleotide with thio-modification, which will lay the foundation for the quality control of antisense drugs and new drug research and development. Methods: Select a target gene containing antisense oligodeoxynucleotide to be tested as a template, and use the thio-modified antisense oligodeoxynucleotide to be sequenced as the downstream primer, and design a primer in its upstream to carry out PCR amplification so that the antisense sequence to be sequenced is incorporated into the PCR amplification product in a modified form; the aforementioned PCR amplification product incorporating the antisense sequence is used as a template, which is automatically sanitized by Sanger dideoxy chain termination sequencing Sequence analysis. Results: The method can detect the antisense sequences with different length of thio modification, and can effectively detect the mutations and deletions generated in the synthesis process. Compared with mass spectrometry, the instruments and reagents are more popular. The method is easy to master and sequenced Low cost, with less sample. Conclusion: This method can be used to confirm the sequence of sulfo-modified antisense oligonucleotide drugs.