Diffusion, mixing, and associated dye effects in DNA-microarray hybridizations.
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 3.8 mm and 2.6 mm, 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 >7.6 mm 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.
Borden
Jacob R
JR
Deptartment of Chemical and Biological Engineering, Northwestern University, Evanston, IL 60208, USA.
Paredes
Carlos J
CJ
Papoutsakis
Eleftherios Terry
ET
D000465:Algorithms; D001703:Biophysics / Q000379:methods*; D002232:Carbocyanines / Q000494:pharmacology; D046969:Clostridium acetobutylicum / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004058:Diffusion; D015964:Gene Expression Regulation, Bacterial; D015233:Models, Statistical; D009693:Nucleic Acid Hybridization / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015345:Oligonucleotide Probes / Q000737:chemistry; D012313:RNA / Q000737:chemistry; D012680:Sensitivity and Specificity; D015398:Signal Transduction; D013997:Time Factors
D002232:Carbocyanines; D018076:DNA, Complementary; D015345:Oligonucleotide Probes; C089187:cyanine dye 3; C085321:cyanine dye 5; D012313:RNA