Institute of Inorganic Chemistry, University of Vienna, Vienna, Austria.
PLoS One. 2014 Jan 15;9(1):e85605. doi: 10.1371/journal.pone.0085605. eCollection 2014.
Cyanine dyes are commonly used for fluorescent labeling of DNA and RNA oligonucleotides in applications including qPCR, sequencing, fluorescence in situ hybridization, Förster resonance energy transfer, and labeling for microarray hybridization. Previous research has shown that the fluorescence efficiency of Cy3 and Cy5, covalently attached to the 5' end of single-stranded DNA, is strongly sequence dependent. Here, we show that DY547 and DY647, two alternative cyanine dyes that are becoming widely used for nucleic acid labeling, have a similar pattern of sequence-dependence, with adjacent purines resulting in higher intensity and adjacent cytosines resulting in lower intensity. Investigated over the range of all 1024 possible DNA 5mers, the intensities of Cy3 and Cy5 drop by ∼ 50% and ∼ 65% with respect to their maxima, respectively, whereas the intensities of DY547 and DY647 fall by ∼ 45% and ∼ 40%, respectively. The reduced magnitude of change of the fluorescence intensity of the DyLight dyes, particularly of DY647 in comparison with Cy5, suggests that these dyes are less likely to introduce sequence-dependent bias into experiments based on fluorescent labeling of nucleic acids.
菁染料常用于 DNA 和 RNA 寡核苷酸的荧光标记，应用包括 qPCR、测序、荧光原位杂交、Förster 共振能量转移和微阵列杂交的标记。先前的研究表明，共价连接到单链 DNA 5'端的 Cy3 和 Cy5 的荧光效率强烈依赖于序列。在这里，我们表明，两种替代菁染料 DY547 和 DY647，它们越来越多地用于核酸标记，具有相似的序列依赖性模式，相邻的嘌呤导致更高的强度，相邻的胞嘧啶导致更低的强度。在所有 1024 种可能的 DNA 5 mers 范围内进行研究，相对于它们的最大值，Cy3 和 Cy5 的强度分别下降约 50%和 65%，而 DY547 和 DY647 的强度分别下降约 45%和 40%。与 Cy5 相比，DyLight 染料的荧光强度变化幅度较小，特别是 DY647，这表明这些染料不太可能在基于核酸荧光标记的实验中引入序列依赖性偏差。
