If you choose to design your own real-time PCR primers, take into consideration that the efficiency (E) of a PCR reaction should be 100%, meaning the template doubles after each thermal cycle during exponential amplification.
Experimental factors such as the length, secondary structure, and GC content of the amplicon can influence efficiency.
- Amplicon length should be approximately 50–150 bp, since longer products do not amplify as efficiently.
- Primers length should be 18–24 nucleotides. This provides for practical annealing temperatures. Primers should be designed according to standard PCR guidelines.
- Specific primers – primers should be specific for the target sequence and be free of internal secondary structure. Primers should avoid stretches of homopolymer sequences (e.g., poly(dG)) or repeating motifs, as these can hybridize inappropriately.
- Primer pairs should have compatible melting temperatures (within 1°C).
- Primers’ GC content should be 50%. Primers with high GC content can form stable imperfect hybrids. Conversely, high AT content depresses the Tm of perfectly matched hybrids. If possible, the 3′ end of the primer should be GC rich to enhance annealing of the end that will be extended.
- Primer-dimers -Analyze primer pair sequences to avoid complementarity and hybridization between primers.
- Avoid genomic DNA contamination – For qRT-PCR, design primers that anneal to exons on both sides of an intron (or span an exon/exon boundary of the mRNA) to allow differentiation between amplification of cDNA and potential contaminating genomic DNA by melting curve analysis.
- Confirm the specificity of your primers – Perform a BLAST® search against public databases to be sure that your primers only recognize the target of interest.
- Optimal results may require a titration of primer concentrations between 50 and 500nM. A final concentration of 200 nM for each primer is effective for most reactions.
A good reaction should have an efficiency between 90% and 110%, which corresponds to a slope of between –3.58 and –3.10. The use of non-optimal reagent concentrations, and enzyme quality, which can result in efficiencies below 90%. The presence of PCR inhibitors can produce efficiencies of greater than 110%.
Many researchers choose to purchase TaqMan® Assay products – primers and probes for real-time PCR designed using a proven algorithm and trusted by scientists around the world.
With pre-developed TaqMan® Assays you can spare yourself the trouble of doing bioinformatics, your assays will all work at Universal Conditions, and they’ll amplify with 100% efficiency.