Detecting Human Functional Sequences with Microarrays

描述（由申请人提供）：随着人类基因组常染色质部分DNA序列的最近完成，决定细胞和生物体功能的遗传信息是可用的。下一个挑战在于了解如何将基因组序列中的信息与其编码的功能联系起来。该项目旨在完善和实施高效的高通量实验方法，以识别编码人类基因组调控和维护功能的序列特征。具体而言，该项目将旨在设计和构建由1.5kb PCR产物组成的微阵列，覆盖ENCODE项目靶向的30 Mb的整个独特基因组序列。平行地，将建立BAC和其他测序瓦片路径克隆的集合以覆盖瓦片路径BAC子阵列中的靶向区域。同时，我们将优化检测方法，以提供富集来自三种活跃分裂细胞系（代表B细胞、T细胞和成纤维细胞）的特定功能元件的DNA组分。将使用基因组微阵列分析这些DNA片段，以鉴定含有功能元件的DNA片段。这将为1%的物理基因组建立各种功能特性的图谱，特别是复制时间，复制起点的位置，染色质修饰和常见的转录因子结合位点。这些图谱将与基因组序列的其他特性进行比较，包括G+C含量、基因含量、重复序列含量和序列保守性。一旦通过复制和初步分析证明数据可靠，所有数据都将存入ENCODE联合体。

DESCRIPTION (provided by applicant): With the recent completion of the DNA sequence of the euchromatic portion of the human genome, the inherited information that determines cellular and organismal function is available. The next challenge lies in understanding how to connect the information in the genome sequence with the functions it encodes. This project aims to refine and implement efficient high throughput experimental methods to identify the sequence features that encode regulatory and maintenance functions of the human genome. Specifically the project will aim to design and construct micorarrays consisting of 1.5kb PCR products covering the entire unique genomic sequence of the 30 Mb targeted by the ENCODE project. In parallel a collection of BAC and other sequencing tilepath clones will be establish to cover the targeted regions in a tile-path BAC subarray. At the same time we will optimize assays to provide DNA fractions enriched for specific functional elements from three actively dividing cell lines representing B cells, T cells and fibroblasts. These DNA fractions will be assayed using the genomic microarrays to identify DNA fragments containing functional elements. This will establish a map of various functional properties for 1% of the physical genome, specifically replication timing, location of replication origins, chromatin modification, and common transcription factor binding sites. These maps will be analyzed in comparison to other properties of the genome sequence including G+C content, gene content, repeat content, and sequence conservation. All data will be deposited with the ENCODE consortium as soon as it is shown to be reliable by replication and preliminary analysis.