This indicates that there are problems with both the PCR and the solid phase hybridization detection. The solid phase hybridization should be able to detect even faint bands on the gel. Due to the low detection limit of the DIAPOPS, true positive samples may not be visible on the agarose gel ( ). For this reason, there are problems with the DIAPOPS, if the weak PCR bands are not detected.

The best way in which to distinguish between these problems is to test the PCR in a non-coated, blocked, NucleoLink Strip. If the product bands on the agarose gel remain less intense than what was expected, the problem is with the PCR, and not a consequence of the covalent bound primer or the procedure of the binding. In this case, the problem of the PCR may be caused by the following factors:

• General problems with preparation of PCR mix
• MgCl2 concentration in the PCR mix is not optimized for the system
• A special compound added to the PCR mix or a special technique in the PCR is necessary for the PCR process to perform optimally (e.g. Hot-Start, DMSO, glycerol)
• An inhibiting compound is present in the PCR
• Annealing temperature is too high for the primers selected
• Problems with storage and handling of primers and probes (e.g. too many freeze thaw cycles of the same stock solution)

If the PCR bands from the reactions in the uncoated NucleoLink Strips are as expected on the agarose gel, the problem must be an inhibition of the PCR reaction occurring in the coated NucleoLink wells. Inhibition of PCR can be attributed to one of the following problems, which all reside in the washing procedure after coating:

• Omitting the wash after coating of solid phase primer (DP-0058, section 2)
• Omitting the wash with water after the NaOH wash which follows the coating of solid phase primer
• Problems with the general washing procedure

The lack of DIAPOPS signal can be caused by two different problems:

1. The DIAPOPS is totally inhibited, and does not give any signal, even if the PCR works as expected ( ).
2. The DIAPOPS is not working optimally resulting in lower signals than normal. In a weak PCR reaction the decrease in DIAPOPS signal due to the sub-optimal conditions will lower the positive DIAPOPS signal below the background signal ( ).

In order to identify the problems associated with hybridization detection it is important to be sure that PCR is functioning optimally in the NucleoLink Strips as detected by appropriate bands on agarose gels.