Probe concentration and probe labelling in DIAPOPS

  1. Introduction
  2. Biotinylated probes
  3. AP labelled probe
    1. Overnight hybridization
  4. Probe labelled with fluorescent tags
  5. Conclusion

1. Introduction
A number of different labels can be used with the probes in the hybridization detection step ( DIAPOPS Procedure) in the DIAPOPS ( DIAPOPS introduction) technique. The following labels have been tested, in the Nunc A/S Research Laboratory:

  1. Probes labelled with biotin for subsequent detection using a conjugate of streptavidin and either alkaline phosphates (AP) ( Alkaline Phosphatase (AP)) or horse radish peroxidase (HRP) ( Horse-Radish Peroxidase (HRP)).
  2. Probes directly labelled with AP.
  3. Probes labelled with Fluorescein or Texas Red for direct detection of the probes without enzymatic signal amplification of the fluorescent signal.

Radioactively labelled probes and probes labelled with Europium have been tested by external laboratories. Detailed results from these investigations are not presented, but results from the experiments were successful.

2. Biotinylated probes
A biotinylated probe has been used in most of the systems tested with DIAPOPS. An optimization of the probe concentration was carried out to determine a concentration giving the highest possible signal. The optimization was not made to find the lowest concentration yielding a detectable signal in order to minimize the costs. Furthermore, the lowest concentration yielding a sufficient, detectable signal is presumably different from one probe sequence to another and must be optimized for each individual system. The concentration of 50 nM biotinylated probe, suggested here ( DIAPOPS Procedure), has been tested with many different probe sequences, and was found to give good, high signals in all DIAPOPS systems tested ( Systems tested in DIAPOPS).

It is possible to use concentrations lower than 50 nM, but the results will not show the same dynamic difference between samples with different initial template concentrations, and the limit of detection can also be affected negatively. Figure 1 shows data from three different concentrations of biotinylated probes. The results clearly show the difference in both the dynamic range and the limit of detection between the three probe concentrations.


Figure 1: Illustrates the DIAPOPS results as a function of probe concentration and initial template concentration. The results are from hybridization of the same strips with the same solid phase amplicons. The first detection was with 0.1 nM, the second with 1.0 nM and the third with 50 nM of biotinylated probe.

The Strip analyzed in Figure 1, was subsequently rehybridized with a probe concentration of 50 nM. This is described elsewhere ( Figure 1: Rehybridization to the solid phase PCR product).

3. AP labelled probe
The probe used for hybridization detection of the solid phase product in the DIAPOPS can be directly labelled with an enzyme, e.g. alkaline phosphatase (AP). This labelling is more expensive than labelling with biotin, and for this reason it is feasible only if probe concentrations lower than 50 nM also are usable. Figure 2 shows results from a DIAPOPS experiment using three different concentrations of AP labelled probe. The results are from different amplifications, and not from rehybridizations ( Rehybridization to the solid phase PCR product) to the same amplicons, as are the results in Figure 1. The results presented in the Figures 1-3 are all from experiments using 4-MUP ( 4-Methylumbelliferyl Phosphate (4-MUP)) as substrate.


Figure 2: DIAPOPS signal with three concentrations of a probe directly labelled with alkaline phosphatase. The decrease in signal seen with the highest initial concentrations of template was also seen on the agarose gels after analysis of the liquid phase PCR products ( Data from a comparison between DIAPOPS and liquid phase product concentration). This indicates that the decrease is caused by differences in the PCR, not in the performance of the solid phase detection.

As can be seen in Figure 2, a concentration of 50 nM gave the best signal in DIAPOPS, and should therefore be selected. However, as mentioned above, the price for this probe is very high, and at Nunc A/S research laboratory we used a concentration of 5 nm with the probe directly labelled with alkaline phosphatase, which was acceptable, although the dynamic range was lower than with 50 nM.

3 a). Overnight hybridization
Hybridization can be performed overnight in the hybridization detection of the solid phase amplicons ( DIAPOPS Procedure). If overnight hybridization is carried out with a biotinylated probe, the results are identical to the results from a 1-hour hybridization with a biotinylated probe (data not shown). If the probe is directly labelled with alkaline phosphatase, the longer hybridization time gives lower signals due to enzyme instability. In Figure 3, overnight hybridization is compared with a hybridization carried out for one hour with different concentrations of the alkaline phosphatase labelled probe. The comparison was performed with two different hybridization buffers.



Figure 3: Comparison of hybridization for one hour (top) with overnight hybridization (bottom). Variation in hybridization solution and in concentration of the alkaline phosphatase labelled probe. No significant difference between the hybridization solutions was seen, although it appeared as though the mix without Denhardt's solution was somewhat better in the overnight incubation experiment. The increase in signal as a function of probe concentration is obvious, and it is also clear that an overnight incubation is not a good choice if the hybridization probe is directly labelled with alkaline phosphatase.

As seen from the results in Figures 1-3, there is no significant difference in signals from the biotinylated probe and the probe directly labelled with alkaline phosphatase when the same concentration is used. This indicates that a high percentage of the biotinylated probes are recognized by the streptavidin alkaline phosphatase conjugate. Consequently, no gain in performance is seen when using the more expensive directly labelled probes. The advantage of the directly labelled probe is a faster assay. The conjugate binding step can be omitted, and thus about one hour of incubation and handling can be avoided.

4. Probe labelled with fluorescent tags
Two different labels have been tested: Fluorescein and Texas Red. If detection by hybridization can be successfully carried out with these labels, both the conjugate binding step and the enzyme reaction step can be omitted from the procedure, therefore time is saved. However, it was not possible to obtain useful signals with these fluorescent probes, because the concentration of the solid phase product is lower than the lowest concentration needed to give a signal with these fluorescent probes.

5. Conclusion
The optimal concentration to use with a biotinylated detection probe is 50 nM. It is possible to use a lower concentration, but only with loss of dynamic range. If a probe directly labelled with an enzyme is used, a concentration of 50 nM raises the price of the assay substantially. It is possible to use the lower concentration of 5 nM, but only with a decreased dynamic range. It is not feasible to use overnight hybridization with directly enzyme labelled probes, due to a degradation of the enzyme.

Two probes directly labelled with fluorescent tags has been tested. The fluorescent tags was; Fluorescein and Texas red. Neither of the probes could be detected after hybridization, and could therefore not be used for detection of the solid phase product. The reason is most likely to be the relatively low concentration of the solid phase product on the surface.