Biochemical Science Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899, USA.
J Agric Food Chem. 2009 Aug 26;57(16):7221-6. doi: 10.1021/jf901165h.
The total amount of DNA in a preparation extracted from tissues can be measured in several ways, each method offering advantages and disadvantages. For the sake of accuracy in quantitation, it is of interest to compare these methodologies and determine if good correlation can be achieved between them. Different answers can also be clues to the physical state of the DNA. In this study, we investigated the lack of correlation between ultraviolet (UV) absorbance and fluorescent (PicoGreen) measurements of the concentration of DNAs isolated from plant tissues. We found that quantitation based on the absorbance-based method correlated with quantitation based on phosphorus content, while the PicoGreen-based method did not. We also found evidence of the production of single-stranded DNA under conditions where the DNA was not fragmented into small pieces. The PicoGreen fluorescent signal was dependent on DNA fragment size but only if the DNA was in pure water, while DNA in buffer was much less sensitive. Finally, we document the high sensitivity of the PicoGreen assays to the detergent known as CTAB (cetyldimethylethylammonium bromide). The CTAB-based method is highly popular for low-cost DNA extraction with many published variations for plant and other tissues. The removal of residual CTAB is important for accurate quantitation of DNA using PicoGreen.
从组织中提取的 DNA 总量可以通过多种方法进行测量，每种方法都有其优点和缺点。为了实现定量的准确性，比较这些方法并确定它们之间是否可以达到良好的相关性是很有意义的。不同的答案也可能是 DNA 物理状态的线索。在这项研究中，我们研究了从植物组织中分离的 DNA 的紫外（UV）吸光度和荧光（PicoGreen）测量浓度之间缺乏相关性。我们发现，基于吸光度的定量方法与基于磷含量的定量方法相关，而基于 PicoGreen 的方法则不相关。我们还发现了在 DNA 没有碎片化的情况下产生单链 DNA 的证据。PicoGreen 荧光信号取决于 DNA 片段大小，但前提是 DNA 处于纯水中，而缓冲液中的 DNA 则不那么敏感。最后，我们记录了 PicoGreen 测定法对去污剂 CTAB（十六烷基三甲基溴化铵）的高灵敏度。基于 CTAB 的方法因其具有成本效益而广泛用于植物和其他组织的低成本 DNA 提取，并且有许多已发表的变体。使用 PicoGreen 进行准确的 DNA 定量时，去除残留的 CTAB 非常重要。
