Optimization of Cyanine Dye Stability and Analysis of FRET Interaction on DNA Microarrays

Abstract

The application of DNA microarrays for high throughput analysis of genetic regulation is often limited by the fluorophores used as markers. The implementation of multi-scan techniques is limited by the fluorophores’ susceptibility to photobleaching when exposed to the scanner laser light. This paper presents combined mechanical and chemical strategies which enhance the photostability of cyanine 3 and cyanine 5 as part of solid state DNA microarrays. These strategies are based on scanning the microarrays while the hybridized DNA is still in an aqueous solution with the presence of a reductive/oxidative system (ROXS). Furthermore, the experimental setup allows for the analysis and eventual normalization of Förster-resonance-energy-transfer (FRET) interaction of cyanine-3/cyanine-5 dye combinations on the microarray. These findings constitute a step towards standardization of microarray experiments and analysis and may help to increase the comparability of microarray experiment results between labs.

An 8-bit monochrome palette of fluorescent nucleic acid sequences for DNA-based painting

“…This larger amplitude (Table S1, ESI†) prompted us to envisage the creation of a colour scale gradient from fluorescently-tagged oligonucleotide arrays at the 3′-end. We chose to focus on Cy3 because of the well-known tendency of Cy5 to photobleach under repeating exposure to 635 nm excitation 28,29 and we started with the complete set of 4 5 permutations. Not only are the positions 4 and 5 nt away from the terminus relevant for fluorescence modulation, 20 but a permutation set of only 3 nucleotides would be insufficient to yield an 8-bit range (4 3 , 64 permutations).…”

Lung Surfactant Accelerates Skin Wound Healing: A Translational Study with a Randomized Clinical Phase I Study

“…Therefore, the arrays were scanned different times with a 4000 B scanner. Images were quantified using GenePixPro 6.0 software and analysed as reported previously [75][76][77] . The gene replicates were tested for outliers with the Nalimov test and the remaining data for each gene were averaged.…”

A Canvas of Spatially Arranged DNA Strands that Can Produce 24-bit Color Depth

“…This phenomenon may be the result of positioning of the red probe on the DNA scaffold, the furthest away from the surface, which affects both DNA synthesis quality and fluorescence intensity due to the significant distance between dye and array surface. Changing the scaffolding order or, alternatively, selecting a more photostable red fluorescent dye 26 should improve performance for this channel.…”