Ditag technologies for complete transcriptome annotation

描述（由申请人提供）：最近完成的人类基因组序列提供了整个遗传信息的框架。然而，基因组内携带的功能内容并没有完全定义。目前的技术不足以完成这项任务。我们的目标是开发一种新的测序策略，以确定完整的转录单位和人类基因组中的顺式调控元件。该策略的概念是将全长转录物的短5'和3'标签提取到双标签结构中用于有效测序。这种双标签测序策略具有准确和有效地映射基因组中所有全长转录本的潜力。当与染色质免疫沉淀（CHIP）技术结合时，这种方法还可以帮助定位转录因子的全基因组顺式调节元件。这项建议的具体目标是： 1)开发一个强大的双标签测序策略，以映射人类基因组中的所有转录单位。我们已经开发了一套原型方法，提取和映射的5'和3'标签的转录序列的基因组。我们将进一步完善ditag stratagy的方法，使其成为完整转录组表征和基因组注释的强大平台。 2)扩大双标签测序的能力，用于转录因子结合位点的全基因组定位。双标签测序的概念直接适用于分析任何DNA片段。我们将开发一个开放的系统来分析ChIP富集的DNA片段的转录因子结合位点的全球定位。 3)在条件p53系统中验证双标签测序方法。我们试图确定所有的基因，是响应p53，并确定通过GIS-ChIP克隆，并通过我们的新GIS分析策略，所有可能的顺式调控片段。

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.