基于毛细管凝胶电泳的寡核苷酸纯度分析方法

doi:10.3969/j.issn.1000-1158.2023.03.13

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摘要在寡核苷酸的化学合成过程中,会产生与全长序列相差一个(n-1)和两个核苷酸(n-2)的杂质,因为分子质量及电荷性质与主成分差异小,难以准确定量检测。毛细管凝胶电泳具有样品用量少、速度快、灵敏度高、分离效果好、自动化程度高等优势,而普遍应用于核酸的分离和分析。采用毛细管凝胶电泳对寡核苷酸纯度进行分析,能有效分离寡核苷酸全长序列与n-1的杂质,在0.03~1.00mg/L浓度范围内线性良好(R2=0.9988),检出限约为0.01mg/L(信噪比S/N>3),该方法的建立将为寡核苷酸的纯度分析与质量控制提供有力的技术支撑。

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关键词 ：计量学, 毛细管凝胶电泳, 寡核苷酸, 纯度分析, 单核苷酸, 分辨率

Abstract：During the chemical synthesis of oligonucleotides, the key technical challenge at present is that the full-length sequence is generated with single (n-1) and double (n-2) deletions as the major impurities. Due to the only subtle difference between the molecular mass and charge properties of impurities and the full-length sequence, it is difficult to accurately quantify the purity of oligonucleotides. With the advantages of small sample amount, fast speed, high sensitivity, separation effective, and high degree automatization, capillary gel electrophoresis is widely used in the separation and analysis of nucleic acids. Capillary gel electrophoresis was used to analyze the purity of oligonucleotides, which can effectively separate the full-length sequence of oligonucleotides and n-1 impurities. The linearity is good in the concentration range of 0.03 mg/L to 1.00mg/L (R2=0.9988), and the detection limit is about 0.01mg/L (S/N>3).The establishment of the aboved method will provide strong technical support for the purity analysis and quality control of oligonucleotides.

Key words： metrology capillary gel electrophoresis oligonucleotides purity analysis single nucleotide resolution

收稿日期: 2022-05-05 发布日期: 2023-03-08

PACS:

TB99

基金资助:上海市市场监督管理局科技项目(2019-12;2022-12)

通讯作者: 刘刚(1982-)男,山东青岛人,上海市计量测试技术研究院正高级工程师,主要从事生物计量与标准方面的研究工作。Email: liug@ simt.com.cn E-mail: liug@simt.com.cn

作者简介: 王乐乐(1990-)男,江苏淮安人,上海市计量测试技术研究院工程师,主要从事生物计量方面的研究工作。Email: wangll@ simt.com.cn

引用本文:

王乐乐,张晓霞,刘刚. 基于毛细管凝胶电泳的寡核苷酸纯度分析方法[J]. 计量学报, 2023, 44(3): 410-414.
WANG Le-le,ZHANG Xiao-xia,LIU Gang. Purity Analysis of Oligonucleotides Based on Capillary Gel Electrophoresis. Acta Metrologica Sinica, 2023, 44(3): 410-414.

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http://jlxb.china-csm.org:81/Jwk_jlxb/CN/10.3969/j.issn.1000-1158.2023.03.13 或 http://jlxb.china-csm.org:81/Jwk_jlxb/CN/Y2023/V44/I3/410

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