What are the properties of a primer?
Posted February 17, 2023
The ideal PCR primer should have certain properties for the reaction to be successful:
- The ideal primer length is between 18 and 30 nucleotides. Shorter primers generally bind or anneal more efficiently to the target segment. However, amplification will not occur if the primer is less than 18 bases in length. Primers longer than 25 bases result in nonspecific amplification.
- Optimum GC content is around 40% - 60%. The GC content or the number of Guanine and Cytosine bases in a selected DNA sequence has a direct impact on amplification. Using primers with higher GC content increases the odds of non-specific amplification and also unnecessarily increases the annealing temperature.
- Annealing temperature must be between 50ºC and 62ºC. Too high annealing temperatures result in no amplification and too low annealing temperatures lead to nonspecific amplification. In addition, the melting temperature difference between forward and reverse primer should be less than 5ºC.
- There should be no complementary region between forward and reverse primers. It’s important that every primer has a unique sequence and that the forward and reverse primer do not match with each other for any assay. If both have a few complementary bases, they will anneal with each other, resulting in the formation of primer-dimers instead of PCR products.
- Primers should not have repetitive bases or complementary bases within the sequence. When primers have repetitive or complementary bases within the sequence, they bind within and form a hairpin-like structure that makes the primer unavailable for amplification.