Primers. General design rules
Primers are short, single-stranded oligonucleotides that are complementary to the target DNA sequence and serve as the starting point for DNA synthesis via DNA polymerase (e.g., Taq polymerase).
The detailed design of primers depends on the aim of the PCR method used (e.g., for mutagenesis, reamplification of plasmids with the introduction of the restriction sites, species differentiation, and genotyping, etc.). For this reason, only general rules of primer design will be given below.
1. The forward primer must be identical to the aiming (sence) strand, and the reverse primer must be identical to the complementary (antisence) strand. Note that the direction of the primer always goes from the 5′-end to the 3′-end, and DNA strands are antiparallel to each other.
2. The melting temperature of flanking primers should not differ by more than 5° C and should be within the range of 55-65 °C. For specific annealing of primers that have similar nucleotide content, it is sufficient to have a difference of two outermost nucleotides in a row at the 3′-end to differentiate between them.
3. Sequence length of primers should be 16-30 nucleotides (nt), preferably with GC content in the 40-60 % range.
4. It is preferable, where it is possible, to have two dGs or dCs per five nucleotides at the 3′-end. Avoid terminal T at the 3′-end.
5. Avoid three or more identical nucleotides in a row and/or repetitive dinucleotides, if it is possible.
6. Please check dimer and hairpin formation.
7. The values of ΔG must be higher than -10 kcal/mol; the values below that number could be the marker of the formation of stable complexes, which might affect the PCR results.
8. For evaluation of annealing temperature, please use software based on the nearest neighbor algorithm. Please note that calculated values of annealing temperature must be below 5 °C of calculated melting temperatures of primers. It is important to test your primers at some range within the calculated annealing temperature.
Probes. The hydrolysis (e.g., TaqMan®) probes design rules
The hydrolysis (e.g., TaqMan®) probes are oligonucleotides, designed to bind between the forward and reverse primers, and contain a fluorophore and a quencher attached to different ends, and possess no secondary structure. Those probes bind to DNA between primer binding sites. While the probe is intact (the fluorophore remains attached to the quencher via the nucleotide sequence), no fluorescence is observed. Taq polymerase hydrolyzes the probe during PCR and releases the fluorophore and quencher into the work solution due to its 5′-3′ exonuclease activity. Thus, the fluorescence signal can be detected, and its level is increasing at each cycle of PCR, and it is proportional to PCR product formation.
General design rules
1. The probe binding site must be in between binding sites of primers. The distance between the probe and primer is not so important, and they might only be one nucleotide apart.
2. Probe melting temperature should be 7-10 °C higher than melting temperatures of primers. It is important for effective probe annealing to target.
3. Probe length should be 18-30 nt with GC content around 30-80 %.
4. Please avoid attaching a fluorophore next to G, as the G has the ability to quench fluorophore signaling.
5. Fluorophore and quencher can be designed at 5′ or 3′ ends, as well as in the middle of the probe sequence via dT.
a. For probes with a length below 25 nt, it is recommended to have terminal modifications.
b. For probes with length higher than 30 nt, it is recommended to place one modification at the 5′ end, whereas another one should be placed in 9-15 nt via dT. Additionally, for this type of probe, it is better to block the 3′ terminal end with phosphate modification, as these probes can also work as primers.
6. It is better to avoid the regions in the probe sequence that are self-complementary (e.g., stem formation domains like in hairpin probes), as these structures might be degraded by polymerase.
Probes. Molecular beacon probes design rules
Molecular beacon probes are oligonucleotides that have only terminal modifications (of fluorophore and quencher) and possess a secondary stem-loop structure (hairpin conformation). The loop domain is a region that is complementary to target DNA (the binding site of this domain must be located in-between binding regions of primers). The stem domain consists of 5-6 GC base pairs that are usually not complementary to the target DNA. In the absence of the target sequence, the complementary regions of the sequence in the stem hybridize, bringing the fluorophore and the quencher into proximity, which leads to quenching of the fluorescence. Binding of the probe to the target sequence leads to the unwinding of its stem-loop structure and an increase in fluorescence intensity. Please note that molecular beacon probes are more susceptible to the presence of non-complementary bases in DNA compared to TaqMan® probes.
General design rules
1. For effective probe annealing, the melting temperature of the loop domain should be 7-10 °C higher than the melting temperatures of primers.
2. The stem domain should consist of 5-8 nt with at least a minimum GC content of 80 %.
3. The sequence of the loop domain should be complementary to the target DNA. The length of the loop domain should be 18-30 nt.
4. The fluorophore is attached to the 5′-end, and the quencher is attached to the 3′-end. Attachment of the fluorophore to a guanine (G) base should be avoided, as it has a quenching effect on fluorophores.
5. The secondary structure of the probe must be checked using DNA folding prediction software. Sequences for which incorrect secondary structures are predicted should be avoided, especially if their ΔG is lower than that of the correct structure.