DNA quantification approach by GE-ICP-SFMS and complementary total phosphorus determination by ICP-SFMS
DNA quantification approach by GE-ICP-SFMS and complementary total phosphorus determination by ICP-SFMS
复制标题
采用 GE-ICP-SFMS 的 DNA 定量方法和采用 ICP-SFMS 的互补总磷测定
DOI：
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发表时间：
2006
期刊：
影响因子：
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通讯作者：
J. Bettmer
中科院分区：
文献类型：
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作者：
Wolfram Brüchert;J. Bettmer
DNA的定量对于许多领域的科学家来说仍然是一个巨大的挑战。在这里，我们提出了用于定量目的的凝胶电泳（GE）和电感耦合等离子体扇形场质谱（ICP-SFMS）的在线耦合。根据目标分析物的 DNA 链长度组成，选择 GE 条件以实现优化分离。特别是，琼脂糖浓度在 0.6-2.2% 范围内变化，对应于从 100 碱基对 (bp) 到基因组 DNA（约 3 Mbp）的 DNA 分离范围。分离的 DNA 化合物以 100 μL min−1 的流速直接输送至 Micromist 雾化器，然后进行 ICP-SFMS，采用 31P 检测，质量分辨率为 4000。对于不同的定量方法，使用 PO43− 标准品和市售 QuantLadder 标准品进行外部校准进行测试。任何分析均可实现低于 3% 的精度：然而，制造商给出的 DNA 浓度指示值并非在所有样品中都能找到。据我们所知，不存在经认证的定量 DNA 参考材料，遗憾的是我们无法证明所开发方法的准确性。尽管如此，我们还是将 GE-ICP-SFMS 耦合应用于基因组 DNA 分析，以确认其分离和检测这些大生物分子的实用性。
Quantification of DNA is still a great challenge for scientists in many fields. Here, we present the on-line coupling of gel electrophoresis (GE) and inductively coupled plasma-sector field mass spectrometry (ICP-SFMS) for quantitative purposes. GE conditions are chosen for optimised separations depending on the target analyte composition in terms of DNA chain length. In particular, agarose concentrations are varied in the range 0.6–2.2%, which corresponds to a separation range of DNA from 100 base pairs (bp) to genomic DNA (approximately 3 Mbp). Separated DNA compounds are directly transported at a flow rate of 100 μL min−1 to a Micromist nebuliser which is followed by ICP-SFMS with 31P detection at a mass resolution of 4000. For their quantification different approaches are tested using external calibration by PO43− standards as well as by a commercially available QuantLadder standard. Precisions lower than 3% can be achieved in any analysis: however, the indicative values for DNA concentrations given by the manufacturers cannot be found in all the samples. As to our best knowledge no certified quantitative DNA reference material exists, unfortunately we could not prove the accuracy of the developed method. Despite this, we then applied the GE-ICP-SFMS coupling to the analysis of genomic DNA in order to confirm its practicability for the separation and detection of these large biomolecules.
影响因子：
2.9
作者：
RYE, HS;DABORA, JM;GLAZER, AN
通讯作者：
GLAZER, AN