HPLC for Separation and Quantification of Deoxyribonucleic Acid Fragments and Measurement of Deoxyribonucleic Acid Degradation
HPLC for Separation and Quantification of Deoxyribonucleic Acid Fragments and Measurement of Deoxyribonucleic Acid Degradation
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用于脱氧核糖核酸片段的分离和定量以及脱氧核糖核酸降解测量的 HPLC
DOI：
10.1007/s10337-014-2723-8
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发表时间：
2014
期刊：
影响因子：
--
通讯作者：
高津章子
中科院分区：
文献类型：
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作者：
柴山祥枝;藤井紳一郎;高津章子
DNA 定量在药物分析和生命科学领域已取得了一定的成果。特别是在核酸药物的质量控制以及转基因生物的检测和定量中，DNA降解率的评估变得势在必行。在本研究中，我们利用阴离子交换柱高效液相色谱法，建立了混合DNA样品中DNA片段的分离和定量方法。通过使用 NaCl 浓度梯度，混合 DNA 样品中的 DNA 片段得到良好分离。获得0.05至12.4ngμL−1的校准曲线，线性良好，相关系数为0.9999。当 S/N = 3 或 S/N = 10 时，检测限为 0.02 ng μL−1，定量限为 0.06 ng μL−1。峰面积重复性测量的相对标准偏差小于 2%。混合 DNA 样品中添加约 1 ng μL−1 的特定 DNA 的回收率为 99.9 ± 3.6%。该方法能够测量 600 bp DNA 的降解率，变化约为 1%。
DNA quantification has made its mark in pharmaceutical analysis and the life sciences. In particular, in the quality control of nucleic acid drugs and the detection and quantification of genetically modified organisms, evaluation of the DNA degradation rate has become imperative. In this study, by using high-performance liquid chromatography with an anion-exchange column, we established a method for the separation and quantification of DNA fragments in mixed DNA samples. By using a NaCl concentration gradient, DNA fragments in mixed DNA sample were separated well. A calibration curve from 0.05 to 12.4 ng μL−1was obtained with high linearity and the correlation coefficient was 0.9999. The limit of detection was 0.02 ng μL−1and the limit of quantification was 0.06 ng μL−1for S/N = 3 or S/N = 10, respectively. The relative standard deviation was less than 2 % in the measurement of peak area repeatability. The recovery of approximately 1 ng μL−1of a specific DNA spiked in a mixed DNA sample was 99.9 ± 3.6 %. The method was able to measure the degradation rate of 600 bp DNA with a variation of approximately 1 %.