This indicates that the PCR performs satisfactorily, but the problem is in the detection of the solid phase product by hybridization ( ). Normally, the DIAPOPS signals are lower than expected in reactions with a high product concentration, which is visualized on the agarose gel, and the signals are absent from reactions with a low product concentration observed on the agarose gel. However, in Nunc A/S research laboratory it has also observed that DIAPOPS signals were high from reactions with a high product concentration, but faded very quickly to become indistinguishable from reactions with a low product concentration, even when PCR bands were visible on agarose gel. This problem is also called "poor sensitivity" ( ).

Poor sensitivity occurs, either because there is a low concentration of solid phase product, or because the hybridization detection is not performing optimally. If it is the first time the batch of coated NucleoLink Strips is tested, the problem could be in the solid phase primer coating procedure. The problem is not in the wash after the coating step as no inhibition of the PCR itself is observed on the agarose gel. The following factors in the coating process can cause the above-mentioned problem:

• There are no or not enough T's added to the 5'-end of the solid phase primer
• The solid phase primer forms primer-dimers with one or both of the liquid phase primers
• The concentration of solid phase primer is incorrect
• Incorrect pH-adjustment of the MeIm buffer in the coating of solid phase primer
• The buffer used in the coating with the phosphorylated solid phase primer is not correct (e.g. TRIS washing buffer with Tween)
• Incorrect concentration of EDC in the binding with the solid phase primer
• Incorrect temperature used in binding with the solid phase primer
• Incubation time used in the covalent binding with the solid phase primer is too short
• The coated NucleoLink Strips are stored at incorrect conditions
• The coated NucleoLink Strips have been blocked with BSA before storage

If the system has not been tested before in DIAPOPS, it is not only the coating which can cause problems. The following factors could also be responsible:

• The primer-ratio in the PCR mix is not optimized for the special system
• The PCR program is not optimized for the DIAPOPS procedure (time, number of cycles)
• The detection probe sequence is not ideal (self-hybridization or looping)
• The hybridization temperature or time is not adequate
• The incubation time and temperature in the incubation with streptavidin-enzyme conjugate are inadequate
• The substrate is not sensitive enough
• The concentration and temperature of the washing buffer used after hybridization are not optimal for the detection probe

If the system and the batch of coated NucleoLink Strips have been tested previously with good results, it is not likely that any of the above mentioned factors is the cause to the problem. A sudden appearance of a problem with a system that normally performs well will most likely come from one of the following factors:

• In the preparation of the PCR mix the primer ratio was made 1:1 (as in standard liquid phase PCR) and not 1:8 (as normally in DIAPOPS)
• Problems with storage and handling of primers and probes (e.g. too many freeze thaw cycles of the same stock solution)
• In the washing step after PCR incorrect concentrations of NaOH or Tween 20 were used
• In the washing step after PCR hot NaOH was used as in the wash after coating
• Insufficient washing after PCR did not denature all solid phase strands, and the solid phase strands may not all have been detected by the hybridization probe
• Incorrect concentration of reagents in the hybridization solution (SSC, Tween 20, blocking reagent, or detection probe)
• Incorrect incubation buffer (e.g. PBS or BSA in buffer) when adding Streptavidin-enzyme conjugate
• The substrate dilution or the substrate stock solution has been stored at an incorrect temperature or for too long
• Incorrect incubation conditions during substrate reaction
• Wrong filters in plate reader are selected (incorrect wavelengths)
• Improper settings of blank values in the plate reader

If there is a possibility that the problem is involved in the detection part of the DIAPOPS procedure, a rehybridization of the same strips is recommended ( ).