Detection of E2F-DNA Complexes Using Chromatin Immunoprecipitation Assays.

TitleDetection of E2F-DNA Complexes Using Chromatin Immunoprecipitation Assays.
Publication TypeJournal Article
Year of Publication2018
AuthorsLee M, Gudas LJ, Saavedra HI
JournalMethods Mol Biol
Volume1726
Pagination143-151
Date Published2018
ISSN1940-6029
KeywordsChromatin, 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).

DOI10.1007/978-1-4939-7565-5_13
Alternate JournalMethods Mol. Biol.
PubMed ID29468550
PubMed Central IDPMC6070307
Grant ListU54 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