Inappropriate/unregulated expression of DNA-binding proteins (DBPs), in particular transcription factors, has been implicated in many diseases, including cancer, AIDS, and diabetes. Current methods for measurement of DBP expression are inadequate for simultaneous measurement of all relevant proteins. The work proposed will provide a genomics-based, parallel method for the measurement of DBP expression using unique genetic tags termed molecular barcodes. Molecular barcodes, developed for the analysis of yeast growth rates, are 20 base DNA sequences designed to minimize cross-hybridization and secondary structure. The barcodes have been synthesized within """"""""cassettes"""""""" with universal forward and reverse PCR primers, allowing single-tube amplification of thousands of cassettes. Using a barcode specific microarray, simultaneous detection of all barcodes within the mixture is possible. With each barcode linked to a particular species within a mixture, detection of the barcode confirms the presence of the encoded species (e.g., the presence of a single strain of yeast within the pool of all possible strains). For the proposed work, new cassettes will be designed that incorporate the recognition sequences of DBPs. Initial experiments on single proteins will validate the protocol for separation of bound and unbound cassettes followed by detection by quantitative PCR. Subsequent experiments using complex mixtures of DBPs and cell extracts will be analyzed using the barcode microarray. The flexibility of application of the barcode cassettes will be demonstrated by simultaneously measuring expression of E. coil, yeast, and human DBP expression on a single microarray. Finally, mutation analysis will be used to determine the most critical bases involved in the DNA:DBP interactions.
| Walton, S Patrick; Mindrinos, Michael N; Davis, Ronald W (2006) Analysis of hybridization on the molecular barcode GeneChip microarray. Biochem Biophys Res Commun 348:689-96 |
