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Development of a method to multiplex ChIP-SEQ and ChIP-chip experiments

开发多重 ChIP-SEQ 和 ChIP 芯片实验的方法

基本信息

批准号：
7802006
负责人：
MICHAEL P WEINER
金额：
$ 9.67万
依托单位：
AFFOMIX, INC.
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-07-01 至 2011-12-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): The immediate objective of our research is to generate a method that will enable researchers to multiplex Chromatin ImmunoPreciptation-Sequencing (ChIP-Seq) analysis in a single Next generation DNA sequencing run. In the to-be-developed method: i) a set of antibodies directed against specific DNA-binding proteins are uniquely bar-coded with a DNA 'ZipCode;' ii) covalent cross-links between DNA-binding proteins and chromosomal DNA are formed by treating cells with formaldehyde; iii) the set of DNA-barcoded antibodies specific to the proteins of interest are used to selectively coimmunoprecipitate the protein-bound DNA fragments that were covalently cross-linked; iv) excess antibodies and chromosomal DNAs are removed by washing; v) the enriched protein-bound chromosomal DNA is ligated to the antibody-attached ZipCode DNA using T4 DNA ligase, and; vi) the immunoprecipitated protein-DNA links are reversed and the recovered DNA is assayed using Next-generation sequencers to determine both the chromosomal DNA sequence bound by the protein and the antibody-identifying DNA ZipCode. PUBLIC HEALTH RELEVANCE: Researchers will benefit from an increased understanding of the function of human proteins. Methods are needed that can facilitate this understanding. The immediate objective of our research is to generate a method that will enable researchers to multiplex the method known as Chromatin ImmunoPreciptation-Sequencing (ChIP-Seq) analysis, in a single DNA sequencing run. We anticipate using our high-throughput antibody-discovery pipeline for producing recombinant antibodies as a means to generate the reagents needed to make this multiplexed method possible.

描述（由申请人提供）：我们研究的直接目标是生成一种方法，使研究人员能够在单个下一代DNA测序运行中进行多重染色质免疫沉淀测序（ChIP-Seq）分析。在待开发的方法中：i)一组针对特定DNA结合蛋白的抗体具有独特的DNA“ZipCode”条形码；（ii） DNA结合蛋白与染色体DNA之间的共价交联是通过甲醛处理细胞形成的；iii)使用一组针对感兴趣的蛋白质的DNA条形码抗体选择性地共免疫沉淀共价交联的蛋白质结合DNA片段；iv)通过清洗去除多余的抗体和染色体dna；v)利用T4 DNA连接酶将富集的蛋白结合染色体DNA与抗体附着的ZipCode DNA连接，并且；vi)将免疫沉淀的蛋白质-DNA链接进行逆转，并使用下一代测序仪检测回收的DNA，以确定蛋白质结合的染色体DNA序列和抗体识别DNA ZipCode。