DNA-arrays (or DNA-chips or microarrays) are flat slabs of glass, silicon or plastic onto which thousands of multiple short single-stranded (ss) DNA sequences (corresponding to small regions of a genome) have been attached. After performing a mRNA extraction in induced and non-induced cells, the mRNA is again reverse transcribed, but here the reaction is tweaked, so that the emerging cDNA contains nucleotides marked with different fluorophores for controls and experiment. Targets will hybridize by base-pairing with those probes that resemble them the most. The array can then be stimulated by a laser and scanned for fluorescence at two different wavelengths (control and induced). The ratio or log-ratio between the two fluorescence intensities corresponds to the induction level.
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
Once the binding motif for a TF is known, this motif (which essentially defines a pattern) can be used to scan sequences in order to search for putative TF-binding site. This is useful, for instance, when trying to identify TF-binding site in ChIP-chip data. Searching for TF-binding site can be done in numerous ways. The most basic method is consensus search, in sequences are scored according to how many mismatches they have with the consensus sequence for the motif. A more elaborate way of searching involves using regular expressions, which allow to search for more loosely defined motifs [e.g. C(C/G)AT]. Common algorithms for this type of search include Pattern Locator and the DNA Pattern Find method of the SMS2 suite, but also some word processors. Finally, the mainstream way of conducting TF-binding site search is through the use of position-specific scoring matrices, which basically count the occurrences of each base at each position of the motif and use the inferred frequencies to score candidate sites. Algorithms in this last category include TFSEARCH, FITOM, CONSITE, TESS and MatInspector.
Quantitative PCR (or quantitative real time polymerase chain reaction) is a modification of the conventional PCR reaction used to amplify and simultaneously quantify a targeted DNA molecule. In its more standard incarnation, a inactive fluorescent dye is added to the PCR mix. This dye is activated upon binding double-stranded DNA; as the PCR iterates, there is more and more dsDNA and therefore more fluorescence intensity.
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.