Development of an Optimal cDNA Array

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DESCRIPTION: (Applicant?s Description) One of the most exciting applications for arrays so far is the monitoring of gene expression (mRNA). Presently, mRNA abundance is determined by hybridizing short or long probes to arrays that are composed of either short sequences of cDNA, long open reading frames of cDNA, or synthesized oligonucleotides. Our objective is to determine what microarray system would give the most accurate and meaningful mRNA abundance information. Known limitations to current schemes prompt us to propose new approaches that will be compared systematically to attain the goal of developing a microarray with the strongest and most reproducible signal representative of the transcription being monitored. Two important questions must be addressed. What type of target link gives the strongest and reproducible signal? Which type of target and probe, long or short, will give the truest representation of each gene? In order to accomplish our goal, the surface and coupling chemistry that give the most immobilized and accessible probe will be determined. The length of probe cDNA that accurately represents an mRNA of interest must also be identified. The working hypothesis is that longer pieces of cDNA will most accurately represent an mRNA because s the length increases there will be less homology between the sequence. Finally, it will be determined if the strongest, most specific hybridization signal is given by the optimal target or a digestion of the optimal target. The model systems parameters will then be used to create an array capable of monitoring mRNA of biological interest, to validate the reproducibility and dependability of our model. This work is innovative because if one microarray could be shown to be superior, scientists can work within one "operating system" to allow communication of expression data in a relevant and meaningful fashion between different experiments and different laboratories.