This problem indicates that the detection of the solid phase products by hybridization is functioning as expected because the signals are in correlation with the PCR itself. This problem can be due to either a minor inhibition of the PCR reaction, or a suboptimal PCR.

Optimization is always necessary for a PCR detection assay, but in some diagnostic systems a special or further optimization of the PCR is needed in order to get an optimal PCR reaction in the coated NucleoLink Strips. The optimal PCR in the coated NucleoLink Strips is very important in order to get good DIAPOPS results. Factors affecting the optimal performance of the PCR are listed below:

• 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)
• Annealing temperature is too high for the primers selected
• The PCR program is not optimized for the DIAPOPS procedure (time, number of cycles)
• Problems with storage and handling of primers and probes (e.g. too many freeze thaw cycles of the same stock solution)

Inhibition of the PCR can arise either from the coated NucleoLink Strips or from some compound in the added DNA template. Inhibition of the PCR comming from the NucleoLink Strips can either be due to reagents that are not properly washed off after the covalent binding of the solid phase primer ( ), or because the NucleoLink wells were not blocked before PCR.

In order to analyze if a compound in the sample DNA inhibits the PCR, an amplification should be carried out in uncoated, blocked, NucleoLink Strips. If the PCR in uncoated, blocked, NucleoLink Strips also yields a decrease in PCR product concentration when analyzed in an agarose gel, the problem of inhibition is caused by a residue in the sample or template. In this case, an optimization of the sample preparation focused on removing the inhibiting substance should be performed.

• A compund in the sample is inhibiting the PCR

If the PCR analysis in uncoated, blocked, NucleoLink Strips indicates that there is no inhibition, the problem derives from the coated NucleoLink Strips, and is probably caused by one of the following factors:

• Omitting the wash with water after the NaOH wash which follows the coating of solid phase primer
• Omitting the wash after coating of solid phase primer (DP-0058, section 2)
• Problems in the general washing procedure
• Coated NucleoLink strips are not blocked with BSA before PCR mix is added