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MICROGENOMICS, How to find binding regions for transcriptional factors? -…
MICROGENOMICS
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INTACT: it isolates nuclei marked with a nuclear membrane protein. We can select and get out the nucleus of interest that express the gene we want. Only the nuclei of interest that are in that cell type are marked and the others not.
LEICA LMD
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Contant and contamination free-method for isolating specific single cells or entire areas of tissue from a wide variety of tissue samples
DNA Footprinting
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Transcription factors and associated proteins that bind promoters, enhacers or silencers to drive or repress transcription -> DNA footprinting helps to elucidate which proteins bind to these associated regions of DNA and unravel the complexities of transcriptional control
- PCR amplify and label region of interest that contains a potential protein-binding site, ideally amplicon is between 50 and 200 bp
- Add protein of interest to a portion of the labelled template DNA; a portion should remain separate for later comparison
- Add a cleavage agent to both portions of DNA template
- A protein that specifically binds a region within the DNA template will protect the DNA it is bound to from the cleavage agent
- Run both samples side by side on a polyacrylamide gel electrophoresis
- The portion of DNA template without protein will be cut at random locations, and thus when it is run on a gel, will produce a ladder-like distribution. The DNA template with the protein will result in ladder distribution with a break in it, the "footprint", where the DNA has been protected from the cleavage agent
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Cleavage agents: DNase I, Hydroxyl radicals, ultraviolet irradiation
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SELEX
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- We take a random DNA oligos
- We can do immunoprecipitation or gel mobility assay
- We purify and amplify by PCR
- We feed that in the next run (iterativity)
- We enrich more and more the binding site
- We get as output a cartoon
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