An DNA amplification
))1. Introduction
An DNA amplification ))
Generally, primers which are performing well in standard liquid phase PCR will also perform well in DIAPOPS.
2. Target sequence
The target DNA sequence should be selected to have the maximum possibility of being unique for the organism analyzed. A good selection is a gene for a virulence factor when analyzing pathogenic bacteria (Rasmussen et al. 1994).
3. Length of PCR product
The selection of a target sequence of 200-400 base pairs (bp) yields the most efficient DNA amplification. It is more difficult to chose primers for longer products. In a PCR designed for specific detection, the maximum efficiency will be gained if the product length does not exceed 1000 bp (Dieffenbach et al. 1993).
4. Specificity of the sequence selected
The primer sequences selected must be checked against the sequence from which they are selected to ascertain whether any non-specific hybridization can occur. This control of the sequence can be carried out using a computer program. In general, the more sequences which are available for analysis, the better the chance of finding the optimal primer pair which only amplifies the correct selected target sequence.
5. Primer length
Normally, primers of 17-28 bases are used. Long primers can use higher annealing temperatures, and may be more specific, but less efficient, because thermodynamically they anneal more slowly (Dieffenbach et al. 1993). There is no requirement for difference in length between the primers selected for standard liquid phase PCR and primers selected for DIAPOPS )
6. Minimal primer-dimer formation
The primers should be selected so as to minimize self-complementarity and 3'-end overlapping in order to minimize primer-dimer formation. Primer-dimer formation may act as a competitor to amplification of the target DNA (Rychlik, 1993). Primer-dimers are synthesized when one primer hybridizes to the other primer and creates a template for the heat-stable DNA polymerase. The primer-dimers can be seen as unclear bands of low molecular weight on the agarose gel analysis of the liquid phase PCR products. In DIAPOPS it is more important to select primers with minimal primer-dimer formation compared to standard liquid phase PCR. This is because DIAPOPS utilizes a solid phase primer )
7. Melting temperature
A high and uniform melting temperature (Tm) of the primers is important to avoid unspecific annealing of one primer at a lower temperature demanded by the other primer. The Tm of the primers can be roughly calculated using the formula (4 x G/C% + 2 x A/T%) (Innis and Gelfand, 1990). Although this formula only strictly applies to primers under 20 bases in length, it is also useful in the evaluation of primers between 20-30 bases. Furthermore, a usefull general rule is that the primers should have a GC/AT ratio similar to or higher than that of the sequence to be amplified (Rychelik, 1993).
8. Hybridization of the 3'-end of the primer
Hybridization of the 3' end of the primer is extremely important in the amplification because the elongation occurs at this end, and it is suggested that G's and C's are selected here to gain the highest number of bindings (Dieffenbach et al. 1993). However, it is also hypothesized that since the 3'-end is so important for the hybridization, the oligonucleotides should not be chosen with G's and C's in this end, as there will be too high a risk of their recognizing false templates and generating false positive results (Rychlik, 1993). Both ideas are plausible, and the only way to select the best sequence in the 3'-end is to optimize it for the individual assay. The terminal oligonucleotide in the 3'-end is also essential for controlling mispriming. If the primers are selected to amplify from sequences where mismatches are present inside the primer recognition site, the 3'-end should be selected at a place of total homology.
9. Self-complementarity
In general, primers that form duplexes, either between primers or internally as hairpin loops, should be avoided. However, if the complementarity does not create any 3'-end terminal duplex, which can be recognized by the polymerase and form primer-dimers, and if the duplex is not too stable, it does not significantly affect the PCR )
10. A linker on the solid phase primer in DIAPOPS
After selection of the PCR primer sequences, one of the primers must be selected as the solid phase primer in DIAPOPS )))
11. Conclusion
There is no difference in the criteria for selection of primers between DIAPOPS and standard liquid phase PCR. As mentioned above, the solid phase DNA amplification is more affected by primer-dimer formation involving solid phase primer, but when the primers are selected with the normal preference of minimal primer-dimer formation, this will represent no problem. In addition, there is no special requiement as to the length of the solid phase primer because a linker is added to ensure that the whole solid phase primer sequence can participate in the reaction )