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Incorporation of Physico-Chemical Parameters Into Design of Microarray Experiments
Ratushna, Vladyslava G.
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Microarrays containing long oligonucleotides provide sensitive and specific detection of gene expression and are becoming a popular experimental platform. In the process of designing an oligonucleotide microarray for Brucella, we optimized the overall design of the array and created probes to distinguish among the known Brucella species. A 3-way genome comparison identified a set of genes which occur uniquely in only one or two of the sequenced Brucella genomes. Reverse transcriptase PCR assays of over one hundred unique and pairwise-differential regions identified in Brucella revealed several groups of genes that are transcribed in vivo with potential significance for virulence. The structural and thermodynamic properties of a set of 70mer oligonucleotide probes for a combined B. abortus, B. melitensis and B. suis microarray were modeled to help perform quantitative interpretation of the microarray data. Prediction and thermodynamic analysis of secondary structure formation in a genome-wide set of transcripts from Brucella suis 1330 demonstrated that properties of the target molecule have the potential to strongly influence the rate and extent of hybridization between transcript and an oligonucleotide probe in a microarray experiment. Despite relatively high hybridization temperatures used in the modeling process, parts of the target molecules are predicted to be inaccessible to intermolecular hybridization due to the formation of stable intramolecular secondary structure. Features in the Brucella genomes with potential diagnostic use were identified, and the extent to which target secondary structure, a molecular property which is not considered in the array design process, may influence the quality of results was characterized.
- Masters Theses