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IDENTIFYING A GENE REGULATED BY A TRANSCRIPTIONAL FACTOR - Coggle Diagram
IDENTIFYING A GENE REGULATED BY A TRANSCRIPTIONAL FACTOR
ChIP
It's an immunoprecipitation, based on finding the binding site
We take the tissue and incubate it, proteins are crosslinked to the DNA.
We isolate the chromatin and shear it in pieces, the proteins are crosslinked thus they remain linked.
We then use antibodies for our protein of interest.
We immunoprecipitate and pull it down.
We then use a protease and purify the DNA
We could then take primers and do a PCR. By RT-PCR we can quantify the amount of the wanted DNA.
Criteria to choose the genes
: i have seen induction or decreasing, some variation on expression; it must have putative binding site; expression profile.
ChIP on chip
In this case we want to use the chip to identify the gene and we do the analysis of the whole genome
We use a
tailing array
Proteins that bind to chromatin are cross-linked in vivo, usually via fixation with formaldehyde
The chromatin is then fragmented and exposed to antibodies specific to the protein of interest
These complexes are then precipitated
The DNA is then isolated and purified
With traditional microarrays, the immunoprecipitated DNA is hybridized to the chip, which contains probes that are designed to cover representative genome regions
ChIP-SEQ
It is a method used to analyze protein interactions with DNA
It is used primarily to determine how transcription factors and other chromatin-associated proteins influence phenotype-affecting mechanisms
It's an alternative to ChIP-chip which require hybridization array, which introduce bias, as an array is restricted to a fixed number of probes