Abstract
The fluorescent intensity of Cy3 and Cy5 dyes is strongly dependent on the nucleobase sequence of the labeled oligonucleotides. Sequence-dependent fluorescence may significantly influence the data obtained from many common experimental methods based on fluorescence detection of nucleic acids, such as sequencing, PCR, FRET, and FISH. To quantify sequence dependent fluorescence, we have measured the fluorescence intensity of Cy3 and Cy5 bound to the 5′ end of all 1024 possible double-stranded DNA 5mers. The fluorescence intensity was also determined for these dyes bound to the 5′ end of fixed-sequence double-stranded DNA with a variable sequence 3′ overhang adjacent to the dye. The labeled DNA oligonucleotides were made using light-directed, in situ microarray synthesis. The results indicate that the fluorescence intensity of both dyes is sensitive to all five bases or base pairs, that the sequence dependence is stronger for double- (vs single-) stranded DNA, and that the dyes are sensitive to both the adjacent dsDNA sequence and the 3′-ssDNA overhang. Purine-rich sequences result in higher fluorescence. The results can be used to estimate measurement error in experiments with fluorescent-labeled DNA, as well as to optimize the fluorescent signal by considering the nucleobase environment of the labeling cyanine dye.
Highlights
The fluorescence of molecules is always sensitive to environmental conditions, the magnitude of changes in the fluorescence intensity of any particular fluorophore depends on its specific modes of interaction with its environment.[1]
Our experiments indicate that the fluorescence of Cy3 and Cy5 is somewhat greater on dsDNA; the differences between our results and previously published results,[15] which show a 2-fold greater fluorescence of Cy3 on ssDNA, may be due to the particular choice of cyanine dye
The results can be used in the planning and analysis of experiments based on the labeling of DNA with cyanine dyes
Summary
The fluorescence of molecules is always sensitive to environmental conditions, the magnitude of changes in the fluorescence intensity of any particular fluorophore depends on its specific modes of interaction with its environment.[1]. The optical systems of sequencers need to balance dynamic range of detection with throughput, making their throughput sensitive to dyes with significant variations in fluorescence.[23] Even though our fluorescence data are obtained on microarrays, most genomics microarray data is fairly insensitive to sequence-dependent fluorescence because the labeling is typically based on reverse transcription using labeled random primers or other quasi-random methods.[24] gene-specific fluorescence intensity effects, due to differences in the relative abundance of nucleobases in particular genes, have been detected.[25] Since both Cy3- and Cy5-labeled single- and double-stranded oligonucleotides are commonly used, we present here comprehensive results for double-stranded DNA to complement and strengthen previous results for Cy3 and Cy5 5′labeled single-stranded DNA.[19] Two types of sequencedependent dye−dsDNA interactions, as illustrated, have been measured: relative intensity of the dyes at the 5′ Two types of sequencedependent dye−dsDNA interactions, as illustrated in Figure 1, have been measured: relative intensity of the dyes at the 5′
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
