DIAPOPS results as a function of a linker in the 5'-end of the solid phase primer

  1. Introduction
  2. A linker on the 5' - end of the solid phase primer
  3. Testing of Poly-T linkers
    1. Number of T's in the poly-T linker
  4. Conclusion

1. Introduction
The solid phase primer used in the DIAPOPS procedure ( Solid phase primer) is covalently bound to NucleoLink via a phosphate group in the 5'-end ( EDC coupling reaction). This makes it possible for the 3'-end to be elongated during PCR ( General PCR introduction). When the solid phase primer sequence is coupled directly to the NucleoLink Strips, there will be very little distance from the side of the wells to the 5'-end of the primer. Therefore, the 5'-end sequence will not be able to participate in the annealing to the liquid phase PCR product, which is the first part of the solid phase DNA amplification ( DIAPOPS). The 3'-end is the part of the solid phase primer which is most important in annealing ( Hybridization of the 3'-end of the primer). However, in order to get the correct specificity in the amplification, most PCR systems are designed to utilize the whole sequence of both primers in the annealing. Therefore, the total sequence of the solid phase primer is significant. Consequently, the ability to use the 5'-end in annealing is important in order to get optimal performance of the amplification. Furthermore, as a consequence of the covalent binding the solid phase primer will not participate as efficiently in the amplification reaction as the liquid phase primers because it cannot move freely.

2. A linker on the 5' - end of the solid phase primer
In order to achieve maximum annealing efficiency of the 5'-end of the solid phase primer, it is important to physically distance the active sequence of the primer from the solid phase. Furthermore, the reaction kinetics of the solid phase primer elongation will be improved by choosing conditions resembling the liquid phase as much as possible. This is also achieved by distancing the active primer sequence from the wall. This will allow the active primer sequence to move more freely enabling it to engage in hybridization and also allow the other reactants of the amplification access to the solid phase primer in a way that resembles the liquid phase primers. The spacing from the solid phase can be achieved by adding a linker to the 5'-end of the solid phase primer between the active primer sequence and the phosphate group used to attach the primer to the NucleoLink solid phase.

3. Testing of Poly-T linkers*
Many linkers are available. The linker used by the Nunc A/S Research Laboratory consists of a number of thymidine (T) bases. This base was chosen as the linker monomer because it is one of the two small bases and because it only uses two hydrogen bonds in the hybridization in the DNA double strand. As a consequence, the risk of false hybridization with the poly-T linker is as low as possible.


Figure 1: DIAPOPS results as a function of the number of T's in the 5'-end of the solid phase primer. The template was a dilution of purified PCR product from a detection of cDNA from potato virus Y (PVY) ( PVY).

Figure 1 shows a comparison of DIAPOPS results from an experiment in which the solid phase primer was both synthesized with a linker of 10 T's and without a linker, binding the 5'-end phosphate group directly to the NucleoLink solid phase. The DIAPOPS signals were clearly improved by the presence of the linker.

3 a). Number of T's in the poly-T linker
When a linker of 10 T's was added to the solid phase primer, the DIAPOPS results were greatly improved, as presented in Figure 1. A linker with 10 T's was chosen because results from previous experiments had shown that 5 T's yielded only minor improvement. When higher numbers of T's were tested in the linker, no further, significant, improvement was seen with any of the systems tested. However, a decrease in the signals has been not been observed using a higher number of T's in the linker, and the linker can contain 20 T's without causing problems.

4. Conclusion
It is very important to have a linker in the 5'-end of the solid phase primer to achieve optimal results in the DIAPOPS. A poly-T linker has been tested with positive results. A linker of at least 10 T's is recommended ( DIAPOPS Procedure).

*Use of a polythymidine linker in solid phase amplification is covered by patents owned by GenSet.