Fluorescent dyes

A range of fluorochromes is available for the labeling of nucleic acids. However, not all fluorochromes are equally suited as labels for oligonucleotides. Newly developed dyes, in particular, should be checked for non-specific staining.

Standard labels for in situ hybridization are the green fluorescein and the red tetramethylrhodamine derivatives. These dyes are well suited for standard applications when the ribosome content of target cells is high. In addition, these dyes can be used in conjunction to label different probes in double staining experiments. CY3 and CY5 are members of the indocarbocyanine family. The high signal intensity of these two dyes make them the fluorochromes of choice when trying to detect small cells with low ribosome content such as bacterioplankton cells. These fluorochromes exhibit high fluorescence due to their high quantum yield and high molar extinction coefficient. It is important to note that the CY5 derivative that fluoresces in the near infrared is detectable only with a CCD camera or on a confocal laser scanning microscope with red laser excitation.

Concomitant to the selection of the fluorescent label, care should be taken that the right optical filters are chosen for the detection of the respective dyes. Well adapted optical filters with a high transmission in the emission spectrum of the dye and a strong and sharp blocking of the excitation light are essential for a confident detection of weak signals.

In environmental samples, single oligonucleotides carrying only one fluorochrome may not provide enough fluorescence signal to detect cells with low ribosome content (Pernthaler et al., 2002). Polynucleotide probes with a length of more than 100 nucleotides carrying several fluorochromes are an alternative (Trebesius et al., 1994; DeLong et al., 1999). However, these probes lack specificity for narrow target groups that are at the level of species and genera.

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Oligonucleotide probes are custom-made by solid-phase synthesis. For detailed information on this process, please refer to the corresponding section of the webpage of our partner biomers.net ( automated oligo synthesis).

In the last step of synthesis a fluorochrome is added to the 5' end of the oligonucleotide. Purified probe stocks are frequently delivered lyophilized. Upon reconstitution with 100 µl sterile water a probe synthesis at 0.02 µmol scale yields approximately a 1,500 ng µl^-1 stock solution.

To determine the exact probe concentration the absorbance of the 1:100 diluted stock solution at 260 nm should be measured, giving 1 OD_{260 nm} ~ 20 ng µl-1 DNA.

 
Furthermore the labeling efficiency of the oligonucleotide should be checked. For a pure monolabeled oligonucleotide the ratio of absorption of the dye (A_dye) versus the absorption of the nucleic acids at 260 nm (A₂₆₀) of a monolabeled oligonucleotide should match the ratio of the extinction coefficients (ε) of the dye and oligonucleotide. The extinction coefficient ε at 260 nm (ε₂₆₀) of an oligonucleotide can be estimated from its nucleotide composition as the sum of the extinction coefficients of the individual nucleotides (dATP = 15.4 cm3 µmol-1, dCTP = 7.3 cm3 µmol-1, dGTP = 11.7 cm3 µmol-1, dTTP = 8.8 cm3 µmol-1). Taking into account the extinction coefficient of the dye (ε_dye) the quality of labeled oligonucleotide can be estimated by calculating a ratio k according to the following formula:

Values of k < 1 indicate an incomplete labeling of a probe, whereas values >1 point to the presence of additional, potentially unbound dye. Considering inaccuracies in the estimation of the extinction coefficients of oligonucleotides, k-values between 0.7 and 1.3 are acceptable.

Working solutions are prepared at concentrations of 50 ng µl^-1 and stored in the dark at -20°C. Prepare only small portions of probe working solutions (50-100 µl). Repeated freeze-thawing may damage the probe and might result in a precipitation of the probe leading to weak hybridization signals and high background.

Lyophilized HRP-labeled probes are suspended in sterile H₂O, too. For calculating the concentration it has to be taken into account that the enzyme itself contributes to the measured absorbance at 260 nm. Therefore the measured A_oligo has to be lowered by a correction factor cf = 0.276. Presuming optimal labeling the peak ratio (A₂₆₀ /A_HRP) should be around 3.

Do not freeze working solutions of HRP-labeled probes, store them in the refrigerator at 4°C! Portions of stock solution can be stored in the freezer, but once an aliquot is thawed keep it at 4°C.