Diffusion, Mixing, and Associated Dye Effects in DNA-Microarray Hybridizations
| Autor: | Jacob R. Borden, Carlos J. Paredes, Eleftherios T. Papoutsakis |
|---|---|
| Rok vydání: | 2005 |
| Předmět: |
DNA
Complementary Time Factors Mixing (process engineering) Biophysics Biology Sensitivity and Specificity Diffusion 03 medical and health sciences Nucleic acid thermodynamics Nucleic Acids Complementary DNA Diffusion (business) Oligonucleotide Array Sequence Analysis 030304 developmental biology Detection limit 0303 health sciences Models Statistical 030302 biochemistry & molecular biology Nucleic Acid Hybridization Gene Expression Regulation Bacterial Carbocyanines Molecular biology RNA Clostridium acetobutylicum DNA microarray Signal intensity Oligonucleotide Probes Algorithms Signal Transduction |
| Zdroj: | Biophysical Journal. 89(5):3277-3284 |
| ISSN: | 0006-3495 |
| DOI: | 10.1529/biophysj.105.067934 |
| Popis: | Typical DNA microarrays utilize diffusion of dye-labeled cDNA probes followed by sequence-specific hybridization to immobilized targets. Here we experimentally estimated the distance typical probes travel during static 16-h hybridizations. Probes labeled with Cy3 and Cy5 were individually introduced to opposite sides of a microarray with minimal convective mixing. Oppositely labeled probes diffused across the initial front separating the two solutions, generating a zone with both dyes present. Diffusion-distance estimates for Cy3- and Cy5-labeled cDNAs were 3.8mm and 2.6mm, respectively, despite having almost identical molecular masses. In separate 16-h hybridization experiments with oppositely labeled probes premixed, arrays that were continuously mixed had 15–20% higher signal intensities than arrays hybridized statically. However, no change was observed in the Cy3/Cy5 signal intensity ratio between continuously mixed and static hybridizations. This suggests that the observed dye bias in diffusion-distance estimates results from differences in the detection limits of Cy3 and Cy5-labeled cDNA, a potential concern for array data on low-abundance transcripts. Our conservative diffusion-distance estimates indicate that replicate targets >7.6mm apart will not compete for scarce probes. Also, raising the microarray gap height would delay the onset of diffusion-limited hybridization by increasing the amount of available probe. |
| Databáze: | OpenAIRE |
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