Team:Glasgow/AzureA

Glasglow

Azure A Staining

Summary

Azure A (Dimethylthionine) is a blue dye of the Thiazin family which can be used to visually stain DNA down to quantities of 20ng. Azure A staining requires no expensive equipment or controlled disposal techniques like the common DNA stain Ethidium Bromide. Glasgow’s 2015 iGEM team utilised Azure A staining for the vast majority of the Agarose gel DNA purification/extractions we performed. We aim to promote Azure A staining in order to expand the participation of community and high school labs in iGEM and molecular biology, where they would not be able to meet the costs of equipment or disposal of Ethidium bromide for DNA visualisation.

Background

Azure A is produced when Methylene blue, another Thiazin stain, is oxidised. Methylene blue is less sensitive for staining DNA than its oxidation products, while Azure A demonstrates reduced background gel staining and a reduced required staining time (NCBE, 2003b). The Thiazin family is thought to stain nucleic acids through ionic interactions with the phosphate groups of the sugar-phosphate backbone (NCBE, 2003a); but weak intercalative interactions with DNA have also been described (Paul and Kumar, 2013).

Figure 2: Chemical structures of four of the Thiazin family of dyes, including Azure A and Methylene blue.

Thiazin dyes are combined with Eosin to make Giemsa stain, a ubiquitous diagnostic stain for intracellular protozoan parasites such as Malaria and Trichomonas. Giemsa stain is also utilised to visualise chromosomal configurations by the so-called “G-Banding” of karyograms, which were the early methods of detecting chromosomal deletions and translocations (Sumner, 1980).
The Azure A compound used herein was Azure A chloride; supplied as a dark green powdered solid available from several chemical supply vendors. The Azure A used by Glasgow Team was purchased from Sigma-Aldrich (A6270)

The costs of staining DNA

Originally, the system containing UirS, UirR, and PlsiR accounts for a negative phototactic response to unidirectional UV-A light. The proposed mechanism puts UirS, a transmembrane protein of the CBCR family, as the molecule that perceives UV light. It is suggested that through a physical interaction between UirS and UirR and possibly a phosphotransfer from UirS to UirR, UirR is released from the transmembrane protein. The released UirR can now bind to DNA and UirR, which is similar to other activators of stress responses, was found to be a transcriptional activator of lsiR after binding to its promoter PlsiR .

Sigma-Aldrich sells 10ml of Ethidium bromide (EtBr) solution at a concentration of 10mg/ml for £45/$56 (E1510) (Sigma-Aldrich, 2015). We used a solution of 15ul (10mg/ml) EtBr per 0.5L of TAE running buffer for post-run staining, which works out to ~6666 0.5L stains per bottle of Ethidium. Traditional DNA staining with Ethidium bromide appears to be low-cost when only the cost of the stain is considered; with a cost per gel of £0.0067. However the cost to a lab is greater than simply the stock stain; Ethidium stained DNA requires visualisation on a UV-transilluminator, and the model of UV transilluminator available to us comes from a range which begins at £600/$900 (VWR, 2015). UV illumination requires an enclosed space where other people and sensitive items will not be damaged by irradiation. Additionally, imaging an Ethidium stained gel requires specialist camera filters to prevent camera sensor damage by the UV radiation, or the use of an enclosed illumination and photographing apparatus such as the BioRad Gel Doc™ XR system. Disposal of Ethidium bromide stained gels is also costly; EtBr has been found to be a potent mutagen in in vitro testing, thus disposal and handling is treated very seriously. The EtBr disposal policy of the University of Glasgow states that EtBr waste should be disposed of into biohazard marked containers, which must be uplifted by a 3rd party waste disposal service.

We have confirmed through the use of a laser scanner that PlsiR is not active when UirS and UirR are absent. PlsiR was ligated to GFP with two ribosome binding sites of different strength and no fluorescence was observed (the parts we used for this experiment were K1725401 and K1725402) (Chart 1). Moreover, cells that possess UirR but lack UirS also did not show levels of fluorescence above the expected for E. coli. Therefore, UirR is not sufficient to drive the activation of PlsiR.

Chart 1. Relative Fluorescence (Compared to Last Taken Measurement of Constitutively Expressed GFP Control) over Absorbance in DH5α cells. DH5α cells containing the PlsiR promoter with GFP fluoresce no more than the original laboratory strain or cells that have GFP without a promoter.

Protocol for producing a 2x stock solution of Azure A stain

We maintained a 2x stock solution in line with the recommendation of the NCBE guidance from which we first learned of Azure A staining (NCBE, 2003b).
To produce 1L of 2x Azure A (0.08% Azure A/40% Ethanol):
• Dissolve 0.8g Azure A chloride solid in 1L 40% Ethanol
• Store in a glass screw capped bottle in darkness, or alternatively wrap the bottle in aluminium foil. Azure A will bleach over time in sunlight and lose effectiveness.
• Prior to use, a 1x (0.04% Azure A/20% Ethanol) solution should be prepared by diluting the 2x stock 1:1 with distilled or deionised water to a desired final volume.
Approximately 500ml of 1x stain was found to be suitable for staining of gels used in our experiments, however this may vary depending on gel and staining tray size.
Similarly 1x should be stored in a glass screw capped bottle in darkness.

Protocol for staining with 1x Azure A

Azure A is used as a post-run gel stain. The effect of putting it in the running buffer is unknown, other than it will definitely stain the gel-kit blue.
• Transfer the gel to a tray or container.
• Glasgow team used a large clear plastic Tupperware box with a lid. A lidded container is preferred to prevent accidental overflowing of the stain over the side.
• Azure A can stain plastics so any tray used for Azure A should not be utilised for other uses unless it has been thoroughly cleaned with 100% Ethanol.
• Pour enough 1x Azure A solution into the container to cover the gel, without overfilling it.
• Shake the container for 10-15 minutes, either by benchtop shaker on a low setting or by hand (fun)
• The 1x stain was shown to be useable for a couple of weeks before making up a new batch, without any demonstrable contamination issues or reduction in effectiveness.
• After a 10-15min stain, strong DNA bands may already be visible at this stage, but we recommend at least one round of destaining for improved visualisation.
• To destain, cover the gel in the used running buffer from the gel-kit, or distilled/deionised water. Shake for 10-15 mins. The destaining solution should be discarded into a sink drain along with running water.
• Repeated rounds of destaining will improve visualisation of weak bands.
• Band visualisation is also improved by viewing the gel on a white lightbox, either inside a clear plastic staining container, or on top of a sheet of acetate to prevent staining of the light box.