Il principio della ChIP: arricchimento selettivo della frazione di cromatina contenente una specifica proteina La ChIP può anche esser considerata una strategia di reverse-genetics su scala genomica
-La qualità dell’anticorpo è fondamentale in questa fase - Deve riconoscere la proteina legata al DNA - Deve essere altamente specifico -Necessaria una pre-taratura per ridurre il legame aspecifico -Gli immunocomplessi sono poi precipitati con ProtG/A Sepharose (lega la regione Fc dell’anticorpo) - lavaggi stringenti riducono il background
DNA-binding proteins are crosslinked to DNA with formaldehyde in vivo. Isolate the chromatin. Shear DNA along with bound proteins into small fragments. Bind antibodies specific to the DNA-binding protein to isolate the complex by precipitation. Reverse the cross-linking to release the DNA and digest the proteins. Use PCR to amplify specific DNA sequences to see if they were precipitated with the antibody.
ChIP seq experiment In Nutshell Protein cross-linked to DNA in vivo by treating cells with formaldehyde Shear chromatin (sonication) IP with specific antibody Reverse cross-links, purify DNA PCR amplification * Identify sequences Genome-wide association map *-unless using a single molecule sequencer
History: From ChIP-chip to ChIP-seq ChIP-chip (c.2000) Resolution (30-100bp) Coverage limited by sequences on the array Cross-hybridization between probes and non-specific targets creates background noise