Tuesday, 1 February 2011

Fluorescent nucleic acid probes

The detection of nucleic acids is important for many life science research tasks, such as sequencing, cDNA synthesis and cloning, RNA transcription and transfection. Accurate detection of DNA/RNA is key to the success of these techniques. DNA/RNA concentration is often measured by UV absorbance at 260 nm. This method, however, is not as accurate and sensitive as DNA quantitation by fluorescent dyes.

The most popular cell-impermeant nucleic stains are 7-AAD, Ethidium Bromide, Propidium Iodide and Stains-all:

7-AAD (7-aminoactinomycin D) is a fluorescent intercalator that binds selectively to GC regions of DNA. 7-AAD/DNA complexes can be excited by argon-ion laser and emit fluorescence with a maxima of 647 nm, making this nucleic acid stain useful for multicolor fluorescence microscopy, confocal laser-scanning microscopy and flow cytometry.
Ethidium Bromide and Propidium Iodide are classic cell impermeant DNA stains that are structurally similar to phenanthridium intercalators. Both dyes exhibit broad absorption in both UV and visible regions, thus they can be conveniently excited with mercury, xenon-arc lamp or argon-ion laser. This makes them suitable for use in a variety of fluorescence instruments such as fluorescence microscopy, confocal laser-scanning microscopy and flow cytometry. EthD-1 and EthD-2 have higher affinity to DNA, RNA and oligonucleotides with a large fluorescence enhancement as compared to ethidium bromide.

Stains-all is a sensitive stain for the electrophoretic characterization of biopolymers on composite agarose-acrylamide gels and hyaluronic acid on polyacrylamide gel. It is suitable for differential staining of nucleic acids and proteins by colour: DNA (blue), RNA (bluish purple) and proteins (red).

The most popular cell-permeant nucleic acid stains are DAPI and Hoechst dyes, such as Hoechst 33342 and Hoechst 33258:

DAPI is an AT-selective minor groove binder. Binding to dsDNA produces a ~20-fold fluorescence enhancement, emits blue fluorescence upon excitation with mercury-arc lamp or UV lines of the argon-ion laser (Ex/Em = 358/461 nm).

DNA-selective Hoechst dyes are classic minor groove-binding dyes. These common nucleus counterstains bind to DNA, and emit blue fluorescence under light excitation (Ex/Em ~ 350/460 nm). They exhibit relatively large Stokes shifts (~110 nm), and are suitable for multicolour labelling experiments. They also exhibit multiple binding modes and distinct fluorescence spectra that are dependent on dye/base pair ratios.

AnaSpec, Eurogentec Group