GENE DISCOVERY BY EPITOPE TRAPPING

描述：(申请者描述)基因改变是基础 许多人类疾病，包括遗传缺陷和癌症。 疾病基因的识别是一个多步骤的、劳动密集型的过程 是基因发现中速度限制的一步。一种快速连接基因的方法 候选疾病间隔中的序列，其中表达的蛋白质 靶组织将有助于识别与疾病相关的基因。 我们提出了一种新的方法(“表位捕获”)，它利用了 从组合文库中进行多样性和选择以快速连接DNA 在基因组的特定区域中的序列，以及在 目标组织。在这种方法中，一个表位文库使用 来自感兴趣的基因组区域的序列(即基因片段)是 针对蛋白质特异性抗体噬菌体展示文库进行筛选 在靶组织中表达。“被困”的表位噬菌体含有DNA 与编码(外显子)区域相对应的序列。捕获的表位 选择噬菌体，对DNA进行测序，并将其映射回基因组区域。一个 这项技术的主要意外之处是快速选择表位特异性 抗体将成为功能分析的强大试剂。我们会 建立表位捕获用于基因发现的验证原理 表达蛋白的5q3l序列及抗体库的构建 造血细胞。具体来说，我们计划：1)产生基因组表位 和细胞特异性抗体噬菌体展示文库，2)评价 表位捕获识别来自5q3l的全序列P1中的已知基因， 3)确定表位捕获是否有效地识别大分子基因 基因组间隔。我们预计，表位捕获与 抗体试剂将允许高通量地鉴定基因 在基因组区域内。

DESCRIPTION: (Applicant's Description) Genomic alterations underlie numerous human diseases, including inherited defects and cancer. Identification of disease genes is a multi-step, labor intensive process and is a rate limiting step in gene discovery. An approach to rapidly link gene sequence in a candidate disease interval with expressed proteins in the target tissue would facilitate identification of disease-associated genes. We propose a novel approach ("epitope trapping") that utilizes the power of diversity and selection from combinatorial libraries to rapidly link DNA sequence in defined regions of the genome with expressed proteins in a target tissue. In this approach, an epitope library, constructed using sequence from the genomic region of interest (i.e. gene fragments), is screened against an antibody phage display library specific for proteins expressed in the target tissue. "Trapped" epitope phages contain a DNA sequence corresponding to a coding (exon) region. The trapped epitope phages are selected, DNA sequenced and mapped back to the genomic region. A major windfall of this technique is rapid selection of epitopespecific antibodies that will be powerful reagents for functional analyses. We will establish proof-of-principle of epitope trapping for gene discovery using 5q3l sequence and an antibodyphage display library for proteins expressed in hemopoietic cells. Specifically, we plan to: 1) generate genomic epitope and cell-specific antibody-phage display libraries, 2) evaluate whether epitope trapping identifies known genes in a fully sequenced P1 from 5q3l, 3) determine whether epitope trapping efficiently identifies genes on large genomic intervals. We anticipate that epitope trapping coupled with antibody reagents will allow for high throughput identification of genes within a genomic region.