Nothing is more frustrating than receiving bad sequence data for the same failing samples. If multiple PCR optimizations have been attempted and the reaction still fails, the likely culprit is bad primers. Primers that are not specific enough for a particular DNA template result in mispriming, or they simply won’t amplify your target. Your best bet is to design new primers. Luckily, this is not as challenging as one might think. Here are some important considerations.
Primer length limitations
Primers are short, made-to-order stretches of oligonucleotides that are synthesized in various lengths. Good PCR primers strike a fine balance between specificity and amplification efficiency. Specificity is controlled primarily by primer length and annealing temperature. For ideal amplification, the best primers are 17 to 24 bases long. The shorter the primers, the more efficiently they can anneal to target DNA. Primers that are longer—say 28 to 35 bases—work better when troubleshooting closely related species, for instance. The longer range allows for higher specificity and room for adding restriction enzyme sites to the primer end, if cloning.
Temperature boundaries
You’ll need to keep in mind that the length and composition of primers directly affects the PCR annealing temperature (Ta). A melting temperature (Tm) of 52°C to 58°C is a good starting range when designing primers. (Longer strands have higher melting temperatures, as do sequences with higher G and C content.) The optimal annealing temperature should be determined empirically, but it is typically lower than the primers’ Tm by approximately 5°C to 10°C. As a rule of thumb, the Tm of the primers can be estimated by adding 2°C for each A or T and 4°C for each G or C. The Tm of a nucleic-acid duplex increases both with its length and with increasing GC content. Make sure the Tm difference of your custom primers is no more than 5°C between the pair.
Determining conserved regions
Armed with these considerations, you need to determine the sequence of the primers—the most important step! You should compare all available related sequences and determine the DNA regions with the least amount of sequence variability using a program like BLAST. These conserved regions serve as the starting places for designing the primers.
When designing primers to amplify DNA from different species, sequences at the 5’- or 3’-untranslated regions of mRNA should be avoided, because they may not have a high degree of homology. The placement of the 3’ end of the primer is critical, in general, for PCR. There should be perfect base pairing between the 3’ end of the primer and the template, minimizing mismatch within the last five to six bases. (It may be tempting to compensate for mismatch by lowering the annealing temperature. However, this rarely works out successfully downstream. You’re better off using degenerate oligonucleotide primers covering all possible combinations for the bases at the 3’ end when detecting sequences with possible variation.) Adding unrelated sequences at the 5’ end is less impactful, as it doesn’t alter the annealing of the sequence-specific portion of the primer.
Avoiding primer-dimers
Another issue with the 3’ primer ends is the possibility of homologies within the primer pair, leading to the dreaded primer-dimer effect. Primer-dimer is when the PCR product obtained is the result of amplification of the primers themselves. This sets up a competitive annealing situation between the template and the primer-dimer product during amplification, negatively affecting results downstream. The risk is greatly increased when multiplexing, and multiple primers are included in the PCR reaction.
Minding GC content
The presence of G and C bases at the 3′ end of the primer—the GC clamp—helps promote correct binding at the 3′ end because of the stronger hydrogen bonding of G and C bases. GC bonds contribute more to the stability—i.e., increased melting temperatures—of primer and template, binding more than AT bonds. Primers with 40% to 60% GC content ensure stable binding of primer and template. However, sequences containing more than three repeats of sequences of G or C in sequence should be avoided in the first five bases from the 3′ end of the primer because of the higher probability of primer-dimer formation.
If you’re new to designing primers, these details may seem overwhelming initially. Don’t despair. There are many primer-designing programs available to help you with the process. Beware that no two primer programs will ever select exactly the same primers, even when basic parameters are equivalently set. If your new primers fail to work, don’t worry. Primers are not expensive, and you can always design more. Practice makes perfect.