1. Introduction
2. DIAPOPS Animation
3. DIAPOPS
   1. Solid phase primer
   2. Asymmetric amplification
   3. Denaturation of solid phase product
   4. Hybridization detection of solid phase product
4. Traditional detection of PCR products
5. Problems with traditional detection of PCR products
   1. Limit of detection
   2. Verification of the amplified product
   3. Differentiation between weakly positive and negative reactions
   4. A high workload
6. DIAPOPS advantages
7. Conclusion

1. Introduction
DIAPOPS is a method for detection of nucleic acids using PCR ( ). In DIAPOPS, the whole analysis from addition of the template to signal detection takes place in the same NucleoLink well. This "Whole in One" procedure facilitates and improves detection of PCR products, especially in the detection of many samples.

2. DIAPOPS animation
The whole DIAPOPS process from template amplification to detection of the solid phase products using hybridization is illustrated in the DIAPOPS animation.

3. DIAPOPS
DIAPOPS means: Detection of Immobilized Amplified Products in a One Phase System. The technique uses a solid phase PCR primer covalently bound ( ) to the wells of the NucleoLink Strips ( ). These primers are elongated during the PCR ( ). This is called a solid phase DNA amplification, and the PCR product are in this way bound covalently to the wells in the NucleoLink Strips. The solid phase products are detected by hybridization ( ).

3 a). Solid phase primer
One of the PCR primers are chosen as the solid phase primer ( ). This primer is synthesized with a linker in the 5'-end, and is furthermore phosphorylated at this end. It is covalently bound to the walls of the NucleoLink wells using carbodiimide condensation between the phosphate group and the NucleoLink surface ( ). Using this approach, the 5'-end is specifically coupled to the surface of the NucleoLink Strips, leaving the 3'-end free to participate in PCR. During the asymmetric amplification in the liquid phase the produced amplicons will recognize the solid phase primers and anneal to these ( ). These newly formed hybrids act as substrates for the heat-stable polymerase in the same way as the liquid phase hybrids. As a result the solid phase primers will be elongated using the annealed products as templates. The elongated products remain attached to the walls of the NucleoLink well via the covalently bound solid phase primers.

3 b). Asymmetric amplification
The aim of DIAPOPS is to produce as many solid phase amplicons as possible; therefore, DNA amplification should be designed to favour the rate of annealing to the solid phase primer. This is achieved by making the amplification in the liquid phase of the DIAPOPS slightly asymmetric. This means that the two liquid phase primers are not added in equal concentrations as in a standard PCR, where the DNA amplification is symmetrical. The concentration of the primer used as solid phase primer is lower than the other primer only found in the liquid phase. As a result the synthesis of products from this other primer is favored. Since these products are present in greater numbers than the other products and do not have as many primers in the liquid phase which they may anneal to, they are more likely to find and anneal to the solid phase primers. This asymmetric amplification greatly enhances the formation of solid phase products ( ).

3 c). Denaturation of solid phase product
The liquid phase amplified products are removed after amplification. The remaining solid phase products are double-stranded, and must be denatured to yield a single-stranded solid phase PCR product. This is carried out by using NaOH ( ). When the solid phase double-stranded products have been denatured and the NucleoLink Strips washed to remove the other strand and excess NaOH, the single-stranded solid phase amplified products are ready for detection by hybridization.

3 d). Hybridization detection of solid phase product
The single-stranded solid phase products in NucleoLink wells are detected by hybridization ( ) using a labelled probe ( ) which recognizes the strand of the solid phase PCR product which is covalently coupled to the surface of the NucleoLink Strips. The label on the probe is detected with an enzyme conjugate, e.g. if the label is biotin, it is recognized by a streptavidin-enzyme conjugate. After removal of excess conjugate a substrate for the enzyme is added ( ). The enzymatic process generates a colored or a fluorescent product, which can be measured in a plate reader.

4. Traditional detection of PCR products
The method normally used to detect PCR products is electrophoresis using a 1% submarine agarose gel. This provides both the size, the number of products of different sizes, and a rough estimation of the concentration of these products. This procedure can supply an answer in 30 minutes if only a few samples are analyzed.

5. Problems with traditional detection of PCR products
There are several problems associated with the traditional method for detection of PCR products. The four main problems when using the agarose gel method for detection of PCR products are:

5 a). Limit of detection
On an agarose gel, five nanograms PCR product is normally necessary for a positive detection, if ethidium bromide is used as the fluorescent agent. This demands a rather high amplification efficiency if only a few, initial template copies are to be detected. Furthermore, if scanning equipment is used to detect and quantify the PCR products ( ), the camera and software are often not able to recognize the faint bands, even if the human eye can see the bands.

5 b). Verification of the amplified product
On the gel, it is only possible to estimate the size of the amplified product. If there is a false amplification of the correct size, there is no control upon the sequence of the product. In order to analyze for the correct sequence, a southern blot is necessary.

5 c). Differentiation between weakly positive and negative reactions
The differentiation between weakly positive samples and negative samples solely depends on the subjective evaluation of a Polaroid photo. Some people will describe a sample giving a very faint band as positive whereas others describe it as negative.

5 d). A high workload
A few samples can rapidly be analyzed on an agarose gel. However, if there are many samples it takes a long time to mix the individual PCR products with load buffer and to load the gel. This is a severe limitation to this detection method in routine use.

6. DIAPOPS advantages
All the problems listed concerning the traditional agarose gel detection method for PCR products, can be solved performing the detection of the PCR products in a MicroWell^® plate format using internal hybridization with an extra enzymatic signal amplification and employing an ELISA reader to analyze the plates and provide the results as figures. These are all solutions incorporated in the DIAPOPS technique using NucleoLink Strips. The main advantages are:

• The extra enzymatic reaction in DIAPOPS improves the limit of detection ( ). Normally, the limit of detection using DIAPOPS is 10-100 times better than normal agarose gel electrophoresis.
• The hybridization detection of the solid phase products adds extra specificity, because the probe is selected internally in the product. Therefore, the specificity is improved as there is only a very small possibility for falsely amplified products to have the correct probe sequence internally.
• When the NucleoLink Strips are measured in an ELISA reader or in a fluorescence plate reader, the results are presented as figures, which can be statistically evaluated. This removes any doubt as to whether a sample should be considered as positive or negative.
• Many samples can simultaneously be tested applying the MicroWell^® plate format. A trained technician can detect the solid phase products in seven NucleoLink plates, a total of 672 samples including controls, in three hours. Furthermore, the analysis has the possibility of being automated, which removes the large workload of the traditional PCR product detection.

7. Conclusion
The DIAPOPS technique uses a solid phase PCR primer covalently bound to the wells in the NucleoLink Strips. These primers are elongated during the PCR, and these PCR products are in this way bound covalently to the wells in the NucleoLink Strips ( ). The solid phase products are detected by hybridization.

Using the DIAPOPS method in NucleoLink Strips, several problems with the agarose gel electrophoresis detection of PCR products are solved. These problems are: Limit of detection, sequence verification of the product, distinction between weakly positive and negative samples, and a large workload when analyzing many samples.