Question

Describe one experimental method used to identify regulatory elements and their domains of regulation (e.g. which tissues are they activated in)

Answer 1

In situ hybridisation utilises labelled cDNA, RNA to isolate specific DNA and RNA sequence in portion or whole tissue. test cells and tissues are typically treated to settle the objective transcripts set up and to expand access of the test. As noted over, the test is either a marked integral DNA or, now most regularly, a correlative RNA (riboprobe). The test hybridizes to the objective grouping at elevated temperature, and afterward the overabundance test is washed away (after earlier hydrolysis utilizing RNase on account of unhybridized, abundance RNA test). Arrangement parameters, for example, temperature, salt, as well as cleanser fixation can be controlled to expel any non-indistinguishable collaborations (i.e., just correct succession matches will stay bound). At that point, the test that was marked with either radio-, fluorescent-or antigen-named bases (e.g., digoxigenin) is limited and evaluated in the tissue utilizing either autoradiography, fluorescence microscopy, or immunohistochemistry, individually.

After cloning cDNA for atranscription factor bacteria is genetically engineered to produce large amounts of protein which further generates antibody. Promoters usually respond to dfferent stimuli in mature tissues or during embryogenesis. Basic promoter analysis can be done by generating promoter deletion mutants. Fragments are then cloned proximal to the reporter gene for e.g lacZ , bacterial gene that encodes b-galactosidase. In the presence of substrate blue color appears. this bacterial protein will be produced wherever the promoter is active. Transgenic mice is generated in which the gene under study is overexpressed in a gven tissue. Gene is then cloned downstream of a tissue specific promoter and introduced into mouse oocytes. Animals which express this gene can be used to study alterations in phenotype.

The most commonly used in vivo method to study for genome-TF binding interaction is based on chromatin immunoprecipitation (ChIP) associated with either DNA microarray technology commonly known as (ChIP-chip) or with massive parallel sequencing (ChIP-seq). , TF–DNA complexes are fixed  by cross-linking with formaldehyde, sheared into pieces with average length of 100–500 bp, and precipitated from solution using a TF-specific antibody. The enriched DNA is measured after reversal of the cross-links by using hybridization to DNA microarrays or deep sequencing