This indicates that the problem is not in the liquid phase DNA amplification ( ), but rather in the detection of the solid phase product ( ). This can either be caused by problems in the covalent binding of the solid phase primer ( ), in the solid phase DNA amplification ( ) or in the subsequent hybridization detection of the solid phase product ( ).

The amount of covalently bound solid phase primers available for elongation ( ) may be decreased or totally absent. This will prevent or decrease the solid phase DNA amplification, and there may be no DIAPOPS ( ) signal. This effect can be caused by one of the following factors:

• The solid phase primer is not 5'-end phosphorylated
• A wrong solid phase primer is used. A solid phase primer either meant for another system, or for the opposite strand which is not recognized by the detection probe
• A different buffer is used with the coating of solid phase primer (e.g. TRIS washing buffer with Tween)
• The EDC was not properly stored before coating with the solid phase primer
• The coated NucleoLink Strips were blocked with BSA before storage

If experiments have shown that there is solid phase primers of the correct sequence on the surface ( ), the problem may also be caused by a poor solid phase DNA amplification in spite of a good liquid phase PCR. This may be caused by the following factor:

• The PCR program is not optimized for the DIAPOPS procedure (time, number of cycles)

If the specific system has not been tested previously in DIAPOPS, a number of factors in the detection by hybridization may also cause the identified problem, This may happen even though there is a good solid phase DNA amplification. These factors are:

• The sequence of the detection probe is designed to anneal to the wrong strand (the strand that is washed off)
• The detection probe is not labelled
• The label on the detection probe is not recognized by the enzyme-conjugate
• The concentration and temperature of the washing buffer used after hybridization are not optimal for the detection probe
• The substrate used is not that required for the selected enzyme
• The substrate is not sensitive enough

If the system has not been tested previously, the problem can most likely be solved by optimization of the conditions in the DIAPOPS procedure ( ). However, if strong bands of PCR products are visible on the agarose gel, it would be expected that some DIAPOPS signals should be present.

If the same batch of coated NucleoLink Strips has been tested previously, and the chosen probes, parameters, enzymes, substrates, and conditions have been successful, it is most likely that the problem lies in the routine handling of the NucleoLink Strips ( ). This may be in one of the following factors which affects the procedure for hybridization detection of the solid phase product:

• The NucleoLink Strips are not properly emptied after BSA blocking
• Insufficient washing after PCR will not denature all solid phase strands, and the solid phase strands may not all be detected by the hybridization probe
• In the washing step after PCR, the strips were not washed with NaOH, e.g. using Tris wash buffer instead of NaOH
• The substrate dilution or the substrate stock solution has been stored at an incorrect temperature or for too long
• Wrong filter set in plate reader is chosen (incorrect wavelengths)
• Problems with storage and handling of primers and probes (e.g. too many freeze thaw cycles of the same stock solution)