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Diffusion, mixing, and associated dye effects in DNA-microarray hybridizations.

ABSTRACT: 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.

SUBMITTER: Borden JR

PROVIDER: S-EPMC1366823 | biostudies-literature | 2005 Nov

REPOSITORIES: biostudies-literature

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Diffusion, mixing, and associated dye effects in DNA-microarray hybridizations.

Borden Jacob R JR Paredes Carlos J CJ Papoutsakis Eleftherios Terry ET

Biophysical journal 20050812 5

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. D ...[more]

PMID: 16100268

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Dataset Information

Diffusion, mixing, and associated dye effects in DNA-microarray hybridizations.

Publications

Diffusion, mixing, and associated dye effects in DNA-microarray hybridizations.

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