Universal Probe Reagents for Detection and Quantitation of RNA Splicing

79744S The number of genes in the genome of higher organisms is relatively small; however, the protein complexity arising from these genes has been shown to be much higher. In part, this is due to alternative splice variants that are created during the processing of gene transcripts. The formation of gene splice variants has implications for human disease (for example, in cancer susceptibility) and also for medical diagnostics, drug development, and bioengineering. However, there are hundreds of thousands of exons for each higher organism, which imposes a technical problem of generating millions of exon specific detection reagents. This project will advance gene splice variant detection and quantitation by utilizing a small library of a few thousand universal-probe reagents and universal-probe microarrays, sufficient to analyze any gene from any organism. Phase I will develop software for the optimal selection of 20 to 50 probe reagents, from the universal library of 1024 reagents, for testing one or more target genes. The efficiency of the overall process will be tested on cDNA clones. Special software functions will be developed to automatically detect skipped exons. The entire procedure will then be tested on several test genes using mRNA prepared from distinct tissues. Lastly, the accuracy of the new splice variant detection and quantification-by-counting method will be evaluated by obtaining full gene sequences of the same set of cDNA clones. Commercial Applications and Other Benefits as described by the awardee: Gene-specific labeled probe cocktails should have many research and diagnostic applications for the detection and quantification of splice variants. Research applications include: fundamental studies of gene expression control, organism development and evolution, understanding disease susceptibility and progression, drug development for gene expression control, and agricultural and industrial research.