Fragmentation of DNA affects the accuracy of the DNA quantitation by the commonly used methods.
Fragmentation of DNA affects the accuracy of the DNA quantitation by the commonly used methods.
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DOI：
10.1186/1480-9222-15-5
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发表时间：
2013-02-13
影响因子：
6.4
通讯作者：
Minarik G
中科院分区：
文献类型：
--
作者：
Sedlackova T;Repiska G;Celec P;Szemes T;Minarik G
具体应用和现代技术，如非侵入性产前检测，非侵入性癌症诊断和下一代测序，目前是全球研究人员关注的焦点。这些在使用高度片段化的DNA分子进行分析时具有共同的特征。因此，对于分子方法的性能，DNA浓度是一个至关重要的参数;我们比较了不同水平的DNA片段化对DNA浓度测量准确度的影响。在我们的比较中，对人工片段化DNA样品测试了目前最常用的DNA浓度测量方法（分光光度法、荧光法和基于qPCR的方法）的性能。在我们的比较中，使用了未片段化和三种特异性片段化的DNA样品。根据我们的研究结果，片段化水平不影响DNA浓度的分光光度测量的准确性，而其他方法，荧光以及qPCR为基础的，显着影响，并观察到更密集的DNA片段化的测量浓度下降。我们的研究已经证实，DNA的片段化水平对三种常用方法中的两种（PicoGreen和qPCR）的DNA浓度测量的准确性具有显著影响。只有分光光度法不受碎片化水平的影响，但该方法的灵敏度是最低的三个测试。因此，如果可能的话，应该使用同样片段化的对照DNA进行DNA定量。
Specific applications and modern technologies, like non-invasive prenatal testing, non-invasive cancer diagnostic and next generation sequencing, are currently in the focus of researchers worldwide. These have common characteristics in use of highly fragmented DNA molecules for analysis. Hence, for the performance of molecular methods, DNA concentration is a crucial parameter; we compared the influence of different levels of DNA fragmentation on the accuracy of DNA concentration measurements. In our comparison, the performance of the currently most commonly used methods for DNA concentration measurement (spectrophotometric, fluorometric and qPCR based) were tested on artificially fragmented DNA samples. In our comparison, unfragmented and three specifically fragmented DNA samples were used. According to our results, the level of fragmentation did not influence the accuracy of spectrophotometric measurements of DNA concentration, while other methods, fluorometric as well as qPCR-based, were significantly influenced and a decrease in measured concentration was observed with more intensive DNA fragmentation. Our study has confirmed that the level of fragmentation of DNA has significant impact on accuracy of DNA concentration measurement with two of three mostly used methods (PicoGreen and qPCR). Only spectrophotometric measurement was not influenced by the level of fragmentation, but sensitivity of this method was lowest among the three tested. Therefore if it is possible the DNA quantification should be performed with use of equally fragmented control DNA.
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