engleski

Tests of rRNA hybridization to microarrays suggest that hybridization characteristics of oligonucleotide probes for species discrimination cannot be predicted

Hybridization of rRNAs to microarrays is a promising approach for prokaryotic and eukaryotic species identification. Typically, the amount of bound target is measured by 3 fluorescent intensity and assumed that the signal intensity is directly related to the target concentration. Using eukaryotic LSU rRNA target sequences and 7, 693 short perfect- 5 match oligonucleotide probes, we have assessed current approaches for predicting signal intensities by comparing Gibbs free energy ( G°) calculations to experimental results. 7 Our evaluation revealed a poor statistical relationship between predicted and actual intensities. Although signal intensities for a given target varied up to 70-fold, none of the 9 predictors were able to fully explain this variation. Also, no combination of different free energy terms, as assessed by principal component and neural network analyses, provided 11 a reliable predictor of hybridization extent. We also examined the effects of single-base-pair mismatch (all possible types and positions) on signal intensities of duplexes. We 13 found that the mismatch effects differ from those that were predicted from solution-based hybridizations. These results recommend against the application of probe-design 15 software tools that use thermodynamic parameters to assess probe quality for species identification. Our results imply that the thermodynamic properties of oligo-nucleotide 17 hybridization are by far not yet understood.

species identification; oligonucleotide microarray; hybridization; mismatch; position; type; purine; pyrimidine; thermodynamics; prediction

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