26th Jan - Transcription II Flashcards
What is ENSEMBL?
A genome browser
How do you create a cDNA library?
Purify RNA
RT into DNA using poly T primers
What are the benefits of cDNA over directly analysing RNA?
More stable than RNA
Can sub-clone
What is a reporter assay?
The gene of interest has been recombinantly modified to include a reporter (e.g. luciferase) that will produce a signal upon transcription.
What is saturation mutagenesis?
Altering each bp individually and measuring the relative activity
What binds to a GC box?
SP1 was found using a gelshift mobility assay
It could also be identified using a promoter mutagenesis experiment
What is a gel shift/ mobility assay?
Use a probe of a DNA region of interest. When the probe is bound to the protein of interest it will be shifted on the gel above the free probe. This can then be labelled with an antibody to create a supershifted band, which can also be flourescently/radioactively identified.
What is the most common MODERN technique for identifying which protein binds to a genetic region of interest?
ChIP-Seq
Outline ChIP -Seq
Used to analyze proteins interaction with DNA
- Cross link cells
- Sonication/mnase digestion
- Digest protein
- Immunoprecipitate
- ChIP library
- Sequence
How was the LCK expression identified to be be driven by TCF1?
LCK Promoter-luciferase assay in HeLa cells showed that LCK had no activity therefore only expressed in T-cells. (SV40 = positive control)
They isolated RNA from different tissues
Seperated the RNA on an agarose gel and blotted
Probed w/ the cDNA sequence which was labelled with 32P
Repeated luciferase activity assay in Jurkat cells, showed high LCK expression
–> Only expressed in T-cells
Analyse sequence of promoter, multiple TCF binding sites show it’s expression is driven by TCF
What is ENCODE?
The encyclopedia of DNA elements, looking for all the binding sites and histone modifications using ChiP Seq
