Il principio della ChIP: arricchimento selettivo della frazione di cromatina contenente una specifica proteina La ChIP può anche esser considerata.

Presentazione sul tema: "Il principio della ChIP: arricchimento selettivo della frazione di cromatina contenente una specifica proteina La ChIP può anche esser considerata."— Transcript della presentazione:

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4 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

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11 - 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

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13 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.

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15 “ChIP” If we have the “right” antibody, we can extract (“immunoprecipitate”) from living cells the protein of interest bound to the DNA And - we can try to identify which were the DNA regions bound by the protein Can be done for transcription factors But can be done also for histones - and separately for each modification

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19 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

20 Workflow of ChIP-Seq

21 Release DNA Immunoprecipitate Sequence Map sequence tags to genome & identify peaks ChIP-seq overview Adapted from slide set by: Stuart M. Brown, Ph.D., Center for Health Informatics & Bioinformatics, NYU School of Medicine DNA + bound protein Prepare sequencing library Fragment DNA

22 ChIP-seq Challenges: Millions of segments Mapping to genome Visualization Peak detection Data normalization …

23 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

24 ChIP-seq big picture Combine high-throughput sequencing with Chromatin Immuno-precipitation to identify specific protein-DNA interactions genome-wide, including those of: Transcription factors Histones (various types and modifications) RNA Polymerase (survey of transcription) DNA polymerase (investigate DNA replication) DNA repair enzymes … or fragments of DNA that are modified (e.g. methylated)

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