This is a weak form of in-silico search, in which the consensus sequence for the motif is compared to genomic positions and the number of mismatches (between candidate site and consensus) is used as a measure of site-quality.
Electro-mobility shift-assays (or gel retardation assays) are a standard way of assessing TF-binding. A fragment of DNA of interest is amplified and labeled with a fluorophore. The fragment is left to incubate in a solution containing abundant TF and non-specific DNA (e.g. randomly cleaved DNA from salmon sperm, of all things) and then a gel is run with the incubated sample and a control (sample that has not been in contact with the TF). If the TF has bound the sample, the complex will migrate more slowly than unbound DNA through the gel, and this retarded band can be used as evidence of binding. The unspecific DNA ensures that the binding is specific to the fragment of interest and that any non-specific DNA-binding proteins left-over in the TF purification will bind there, instead of on the fragment of interest. EMSAs are typically carried out in a bunch of fragments, shown as multiple double (control+experiment) lanes in a wide picture. Certain additional controls are run in at least one of the fragments to ascertain specificity. In the most basic of these, specific competitor (the fragment of interest or a known positive control, unlabelled) is added to the reaction. This should sequester the TF and hence make the retardation band disappear, proving that the binding is indeed specific
In motif discovery, we are given a set of sequences that we suspect harbor binding sites for a given transcription factor. A typical scenario is data coming from expression experiments, in which we wish to analyze the promoter region of a bunch of genes that are up- or down-regulated under some condition. The goal of motif discovery is to detect the transcription factor binding motif (i.e. the sequence “pattern” bound by the TF), by assuming that it will be overrepresented in our sample of sequences. There are different strategies to accomplish this, but the standard approach uses expectation maximization (EM) and in particular Gibbs sampling or greedy search. Popular algorithms for motif discovery are MEME, Gibbs Motif Sampler or CONSENSUS. More recently, motif discovery algorithms that make use of phylogenetic foot-printing (the idea that TF-binding site will be conserved in the promoter sequences for the same gene in different species) have become available. These are not usually applied to complement experimental work, but can be used to provide a starting point for it. Popular algorithms include FootPrinter and PhyloGibbs.
Quantitative Reverse-Transcription PCR is a modification of PCR in which RNA is first reverse transcribed into cDNA and this is amplified measuring the product (qPCR) in real time. It therefore allows one to analyze transcription by directly measuring the product (RNA) of a gene's transcription. If the gene is transcribed more, the starting product for PCR is larger and the corresponding volume of amplification is also larger.
Regulated genes for each binding site are displayed below. Gene regulation diagrams
show binding sites,
both positively and negatively regulated
genes, genes with unspecified type of regulation.
For each indvidual site, experimental techniques used to determine the site are also given.