Primer design is an important step required for PCR experiments. When designing a primer, you will have to consider several components to help you avoid troubleshooting a lot during the process.
Types of Primers
Living organisms use RNA primers where as other invitros often involves DNA primers. But DNA primers are more preferred because of various reasons, including easy storage, stability, and fewer enzymes needed to start the synthesis process. Here are more comparisons between RNA primers and DNA primers:
- Reaction – The process of amplifying DNA primers depend on temperature and fewer proteins, while the replication process is often catalytic reaction that depends on fewer proteins and enzymes.
- Synthesis – RNA primers need Primase enzyme, whereas DNA primers are chemically synthesized.
- Length – RNA primers are have between 10 and 20 base pairs, while DNA primers have base pairs, ranging from 18 to 24.
What Makes a Primer Good?
In primer design, researchers aim for GC content to be 40-60%, with the 3’ of primer ending in C or G to promote binding. That’s what we call a GC Clamp. Both C and G bases have a strong hydrogen bonding and helps with primer’s stability. But try to be careful not to have many repeating C or G bases because this may result in the formation of primer-dimer. Primer design for Polymerase Chain Reaction (PCR) Experiments Another thing is that, the recommended length of a primer makes it good. Ideally, it should be between 18 and 30 bases. Usually, specificity depends on annealing temperature and length. This means shorter primers anneal or bind to the target more efficiently.
Using Software in Primer Design
A couple of tools and solutions are available to help both experienced and new users design primers. These tools minimize the time and cost involved during experiments by reducing the risks of failed experiments. For instance, at Eurofins Genomics, they design primers for restriction analysis of enzymes, alignments, and single templates. But because guidelines for primer design may vary, researchers will need software like Beacon Designer and AlleleID. They are capable of designing oligonucleotide probes and primers for detecting more complex assays, like multiplex assays.
Steps
- Keep primer’s melting temperature within 2°C.
- Use annealing temperature of between 3-5°C.
- Make primers range from 18-22 bases.
- Design with GC content of between 35 and 65%.
- Reduce GC repeats at the 3’ end of primers.
- Limit length of amplicon to about 140 base pairs.
Testing Primers against Sequence
Like designing, testing involves several steps, too. First, you will need to screen for any physical primer-dimers, hairpins, and properties. Second, select every PCR product on the sequence in a pair so as to extract products of your primers. Third, extend primers with polyA tails, restriction sites, or other custom sequences. Lastly, dynamically calculate melting point of DNA for target regions for any manual primer design.
The bottom line is that oligonucleotide primers are important when running PCR reactions. As a researcher, you will have to design primers, which complement to DNA’s template region. You should also ensure they are chemically synthesized by binding several nucleotides and blocking/unblocking reactive groups.
