Assay for the quantification of intact/fragmented genomic DNA
Assay for the quantification of intact/fragmented genomic DNA
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DOI：
10.1016/j.ab.2006.07.035
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发表时间：
2006-11-15
影响因子：
2.9
通讯作者：
Papapostolou, Ioannis
中科院分区：
文献类型：
--
作者：
Georgiou, Christos D.;Papapostolou, Ioannis
这项研究表明，常用的基于 Hoechst 和 PicoGreen 的检测方法对基因组 DNA 进行定量的准确性受到其片段化程度的极大影响。具体来说，结果表明这些测定法低估了 70% 的大小小于 23 kb 的双链 DNA (dsDNA) 的浓度。另一方面，大于和小于约 23 kb 的 DNA 大小通常通过琼脂糖电泳 DNA 涂片测定分别表征为完整和片段化基因组 DNA，并且仅通过该测定进行定性评估。由于需要对片段化和总基因组 DNA 进行准确定量，加上缺乏特异、可靠且简单的定量方法，促使我们开发了一种基于 Hoechst/Pico Green 的荧光测定法，可以对两种类型的 DNA 进行定量。该测定解决了这些问题，在其 Hoechst 和 PicoGreen 版本中，它分别使用 Hoechst 或 PicoGreen 准确定量浓度低至 3 ng ml(-1) 或 5 pg ml(-1) 的完整 (>= 23 kb) 或片段 (< 23 kb) dsDNA，以及浓度低至总 DNA 样品中以任何比例存在的完整/片段 DNA 的各个片段。分别使用 Hoechst 或 PicoGreen 配制 10 ng ml-1 或 15 pg ml-1。由于该测定可区分两个大小范围（>= 23 和 < 23 kb）的总基因组 DNA 并对其进行定量，因此建议将其作为琼脂糖凝胶电泳基因组 DNA 涂片测定的定量替代品。 sigma (c) 2006 Elsevier Inc. 保留所有权利。
This study shows that the accuracy of the quantification of genomic DNA by the commonly used Hoechst- and PicoGreen-based assays is drastically affected by its degree of fragmentation. Specifically, it was shown that these assays underestimate by 70% the concentration of double-stranded DNA (dsDNA) with sizes less than 23 kb. On the other hand, DNA sizes greater and less than approximately 23 kb are commonly characterized as intact and fragmented genomic DNA, respectively, by the agarose electrophoresis DNA smearing assay and are evaluated only qualitatively by this assay. The need for accurate quantification of fragmented and total genomic DNA, combined with the lack of specific, reliable, and simple quantitative methods, prompted us to develop a Hoechst/Pico Green-based fluorescent assay that quantifies both types of DNA. This assay addresses these problems, and in its Hoechst and PicoGreen version it accurately quantifies dsDNA as being either intact (>= 23 kb) or fragmented (< 23 kb) in concentrations as low as 3 ng ml(-1) or 5 pg ml(-1) with Hoechst or PicoGreen, respectively, as well as the individual fractions of intact/fragmented DNA existing in any proportions in a total DNA sample in concentrations as low as 10 ng ml-1 or 15 pg ml-1 with Hoechst or PicoGreen, respectively. Because the assay discriminates total genomic DNA in the two size ranges (>= 23 and < 23 kb) and quantitates them, it is proposed as the quantitative replacement of the agarose gel elect ropho resis genomic DNA smearing assay. sigma (c) 2006 Elsevier Inc. All rights reserved.