Ditag technologies for complete transcriptome annotation

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DESCRIPTION (provided by applicant): The recently finished human genome sequences provide a framework of the entire genetic information. However the functional contents carried within the genome are not completely defined. Current technologies are inefficient to complete this task. Our goal is to develop a new sequencing strategy to define the full transcript units and the cis-regulatory elements in the human genome. The concept of this strategy is to extract short 5' and 3' tags of full length transcript into a ditag structure for efficient sequencing. This ditag sequencing strategy has the potential to accurately and efficiently map all full-length transcripts in the genome. When coupled with the chromatin immunoprecipitation (CHIP) technology, this approach can also help to localize genome-wide cis-regulatory elements of transcription factors. The specific aims in this proposal are to: 1) Develop a robust ditag sequencing strategy to map all transcript units in the human genome. We have developed a set of prototype methods that extract and map the 5' and 3' tag of transcript sequences to the genome. We will further refine the methodologies of ditag stratagy to become a robust platform for complete transcriptome characterization and genome annotation. 2) Expand the capacity of ditag sequencing for genome-wide localization of transcription factor binding sites. The concept of ditag sequencing is directly applicable for analyzing any DNA fragments. We will develop an open system to analyze ChIP enriched DNA fragments for global localization of transcription factor binding sites. 3) Validate the ditag sequencing approach in a conditional p53 system. We seek to identify all genes that are responsive to p53, and to ascertain through GIS-ChIP cloning and through our novel GIS analysis strategy all possible cis-regulatory fragments.