Title | Detection of E2F-DNA Complexes Using Chromatin Immunoprecipitation Assays. |
Publication Type | Journal Article |
Year of Publication | 2018 |
Authors | Lee M, Gudas LJ, Saavedra HI |
Journal | Methods Mol Biol |
Volume | 1726 |
Pagination | 143-151 |
Date Published | 2018 |
ISSN | 1940-6029 |
Keywords | Chromatin, Chromatin Immunoprecipitation, DNA, E2F Transcription Factors, High-Throughput Nucleotide Sequencing, Humans, Polymerase Chain Reaction, Sonication |
Abstract | Chromatin immunoprecipitation (ChIP), originally developed by John T. Lis and David Gilmour in 1984, has been useful to detect DNA sequences where protein(s) of interest bind. ChIP is comprised of several steps: (1) cross-linking of proteins to target DNA sequences, (2) breaking genomic DNA into 300-1000 bp pieces by sonication or nuclease digestion, (3) immunoprecipitation of protein bound to target DNA with an antibody, (4) reverse cross-linking between target DNA and the bound protein to liberate the DNA fragments, and (5) amplification of target DNA fragment by PCR. Since then, the technology has evolved significantly to allow not only amplifying target sequences by PCR, but also sequencing all DNA fragment bound to a target protein, using a variant of the approach called the ChIP-seq technique (1). Another variation, the ChIP-on-ChIP, allows the detection of protein complexes bound to specific DNA sequences (2). |
DOI | 10.1007/978-1-4939-7565-5_13 |
Alternate Journal | Methods Mol. Biol. |
PubMed ID | 29468550 |
PubMed Central ID | PMC6070307 |
Grant List | U54 CA163071 / CA / NCI NIH HHS / United States U54 CA163068 / CA / NCI NIH HHS / United States G12 MD007579 / MD / NIMHD NIH HHS / United States U54 MD007587 / MD / NIMHD NIH HHS / United States R25 GM082406 / GM / NIGMS NIH HHS / United States |