Research Project
7802006
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.
