Primer design tips and tricks

  • Published: 22 September 2021
Whenever you are planning to do a PCR experiment, whether it is end-point PCR, qPCR, RT-PCR, or some other sort of PCR, you are going to need primers. Getting the right primers can be difficult and you can't just go and buy random primers either, you have to design them first. Although for some experiments you can use a random primer mix (usually not for PCR though) or you can ask someone else to design the primers for you. Still doing it yourself will make you feel a great sense of accomplishment, so here is a quick overview of how it is done.
What is a primer?
A primer is a short nucleic acid sequence, which is used as a starting point for DNA replication. Since a primer is single-stranded you will need two for a PCR experiment - one for forward and one for reverse strand. 
Why are primers necessary?
A primer, if designed correctly, should, after binding to template DNA, mark the starting point of the DNA sequence of interest. A primer is also the place where the DNA polymerase binds, so without the primers, the polymerase chain reaction cannot start. 
 
Where is it possible to design primers?
There are many different computer programs for designing primers. The most popular one is definitely Primer-BLAST, but there are many others like Primer3, PrimerQuest, OLIGO, etc., so you can choose what works best for you.
How to design primers?
You would think that using a computer program to design a primer is the hardest part of the whole process, but it actually isn't. It is one of the easiest parts. Getting to that part might take time though. You first need to find the correct template DNA sequence that you want to replicate. Depending on if you want your primers to be variant-specific, species-specific, universal, or something else you have to find the most suitable area on your DNA to replicate. For example to find variants you need an area that is that variant specific, but for universal primers, you need to find an area on your template DNA that is conserved in all species. Finding this correct template DNA region might be a bit of a headache, but once it is done you can enter your chosen sequence into the primer designing program and the program will automatically generate primers for you.
 
Then you will have your primers, but the work doesn't end there. Primers still need to be optimized - you need to find the correct annealing temperature and concentration for your experiment. Also, make sure there isn't a lot of nonspecific binding of primer dimers. Luckily there are programs that can make this step easier for you as well. 
 
If you have done all you can and the experiment doesn't work, then you have to go back to the start and redesign your primers. Ordering primers isn't very expensive, but you might have to redesign them multiple times before you get it right.
 
What to keep in mind when designing primers?
There are many things to keep in mind when designing primers. As previously mentioned, you have to consider how specific you want your primers to be and optimize them. You also have to make sure your primers are compatible with the detection method you are using.
 
Primer designing programs definitely save you from some extra thinking, since they pretty much do the designing for you, but there are certain universal properties that a good primer should have. A primer shouldn't be very long - about 18-26 bp. The GC content should be around 50%. The melting temperature difference between primers shouldn't be over 5°C.  The melting temperature itself should be around 60-65°C. The forward and reverse strand primers shouldn't have any complementary regions, otherwise, they will bind to each other. Also, long runs of a single base like  ATTTTTT or repeats like ATATATAT should be avoided. It would be best to consider these things already while choosing the DNA sequence of interest. 
How are synthetic primers made?
While in nature living organisms use RNA primers, labs use DNA primers, because they are more stable. These synthetic primers are chemically synthesized by joining nucleotides together one by one usually in the 3’ to 5’ direction using phosphoramidites. This process can also have many error sequences (sequences that are not completed/ shorter than required), therefore after the synthesis, the mix has to be purified, so that only the correct primers remain. 
 
What are the differences between designing primers and probes?
Designing primers and probes isn't really that different when it comes to choosing a sequence (length, GC content, base repeats, etc). 
 
The main difference comes from their function. Primers are to replicate your sequence of interest while probes are to detect that sequence. There are different computer programs for designing primers and probes for that reason, but you still have to keep one in mind while designing the other. With probes basically, you only have to think about finding the right location on your template DNA. With probes, you also have to think about reporter fluorophores and quenchers and make sure they match with each other. This is especially important when using multiplexing. 

What to consider when using primers and probes at the same time?

When you are doing probe-based qPCR you have to make sure that your primers and probes are compatible with each other. As previously mentioned you have to make sure their sequences don't overlap but are in the correct position to each other. Also, probes should have a melting temperature of 6-10°C higher than the primers to ensure better sensitivity. The annealing temperature of a primer-probe set should be set a maximum of 5°C below the lower primer melting temperature to allow specific binding. You will probably have to do some extra optimization once you have them both. 
 
What happens next?
Once you have found your sequence of interest and designed primers it is time to experiment! For that, you are going to need a PCR mix or a qPCR mix, which you can find from the Solis BioDyne product list. Don't be afraid to order too much, since making sure that your primers are binding to the correct place, there isn't a lot of non-specific binding, there aren't many primer dimers, the concentration is correct and the temperature optimal may take quite a lot of tries before getting it all right. We've all been there, so we know that at times it can be frustrating, but it will all be worth it in the end.
 
Bustin, S., & Huggett, J. (2017). qPCR primer design revisited. Biomolecular Detection and Quantification, 14, 19–28.doi:10.1016/j.bdq.2017.11.001