Abstract
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.
Highlights
Fluorescent readout from labeled nucleic acids on solid surfaces or in solution is a common element in a broad range of biotechnological and biophysical methodologies
In most cases, such as in microarray experiments, sequencing-by-synthesis, qPCR, and fluorescence in situ hybridization (FISH), the objective is to quantity the abundance of the labeled molecule
Previous experiments have shown that the fluorescence of the cyanine dyes Cy3 and Cy5, which are commonly used in nucleic acid labeling applications, are very sensitive to their nucleobase environment, both to nucleobases in solution [1], and covalently bound to the 59 termini of both single- [2], [3] and double-stranded DNA [4]
Summary
Fluorescent readout from labeled nucleic acids on solid surfaces or in solution is a common element in a broad range of biotechnological and biophysical methodologies. Previous experiments have shown that the fluorescence of the cyanine dyes Cy3 and Cy5, which are commonly used in nucleic acid labeling applications, are very sensitive to their nucleobase environment, both to nucleobases in solution [1], and covalently bound to the 59 termini of both single- [2], [3] and double-stranded DNA [4].
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