高等学校化学学报

高等学校化学学报 ›› 2010, Vol. 31 ›› Issue (12): 2360.

• 研究论文 • 上一篇    下一篇

荧光标记DNA高分辨电感耦合等离子质谱定量分析

高运华1,李海峰1,李建新2,黎朋1,叶健3,赵兴春3   

  1. 1. 中国计量科学研究院
    2. 山东省菏泽市产品质量监督检验所
    3. 公安部物证鉴定中心
  • 收稿日期:2010-07-22 修回日期:2010-09-01 出版日期:2010-12-10 发布日期:2010-12-06
  • 通讯作者: 高运华 E-mail:gaoyh@nim.ac.cn
  • 基金资助:

    国家科技支撑计划项目(批准号: 2008BAK45B04)和国家质检公益专项(批准号: AHY0709)资助.

Quantitative Analysis of Fluorescent Dye\|labeled DNA by High Resolution Inductively Coupled Plasma Mass Spectrometry

GAO Yun-Hua1*, LI Hai-Feng1, LI Jian-Xin2, LI Peng1,YE Jian3, ZHAO Xing-Chun3   

  1. 1. National Institute of Metrology, Beijing 100013, China;
    2. Supervision and Examination Station of Product Quality of Heze City, Heze 274000, China;
    3. Institute of Forensic Science of Ministry of Public Security, Beijing 100038, China
  • Received:2010-07-22 Revised:2010-09-01 Online:2010-12-10 Published:2010-12-06
  • Contact: GAO Yun-Hua E-mail:gaoyh@nim.ac.cn
  • Supported by:

    国家科技支撑计划项目(批准号: 2008BAK45B04)和国家质检公益专项(批准号: AHY0709)资助.

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摘要： 建立了基于磷元素测量的高分辨电感耦合等离子质谱定量荧光标记DNA的分析方法，该方法定量测量结果可以溯源到国际基本单位（SI）。采用柱层析、超速离心、透析的技术对样品进行纯化，用芯片电泳和电导率测试仪对其进行了纯度检验。然后利用优化后的微波消解方法对荧光标记DNA样品进行了消解处理。从射频功率、等离子气流速、辅助气流速、雾化气流速、采样深度、获取时间等方面对高分辨电感耦合等离子质谱测量条件进行了优化，从物理性干扰、内标、同位素、元素形态等方面对测量进行了校正。利用优化后的方法对荧光标记DNA样品进行了定量测量，从方法的精密度、标准物质、样品称量、标准和样品稀释等方面进行了定量测量结果不确定度的评估。测量结果的扩展不确定度为8.28%(k=2)，远优于现在常规的紫外、荧光、色谱测量核酸含量的不确定度。该方法可用于核酸含量标准物质的定值分析。

Abstract: The accurate mass determination method of fluorescent dye-labeled oligonucleotides by high resolution inductively coupled plasma mass spectrometry (HR-ICP-MS) was established. This method allows for the mass of fluorescent dye-labeled oligonucleotides to be traceable to the SI. The fluorescent dye-labeled oligonucleotides was processed by column chromatography、ultracentrifugation、dialysis。The purity was detected by electrophoresis chip and electrical conductivity。The fluorescent dye-labeled oligonucleotides was digested with microwave in nitric acid. The phosphorus from the fluorescent dye-labeled oligonucleotides was quantitative determined by the HR-ICP-MS. The parameters of HR-ICP-MS and microwave digestion was optimized. The interference factors of HR-ICP-MS such as physical interference, signal drift interference, isotopes, phosphorus element speciation interference was analyzed and corrected with ultra-filtration, internal standard of Y（88.906）, certified reference material of phosphate, nucleotide, oligo DNA. The uncertainty in measurement was evaluated mainly from method precision, Phosphorus CRM, Nitric acid digestion, Weight of sample and CRM. A uncertainty (< or = 8.28%) was smaller than ultraviolet spectrophotometry, fluorescence spectrophotometry and real time PCR. This method will aide in the development of Certified Reference Materials (CRMs) with certified nucleic acid（DNA、RNA） content.

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