Primers will have the following characteristics:
- Primers will have a sequence length of 17 to 30 nucleotides. This enables unique annealing of the primer to a single sequence of the genome.
- Their GC content will be at least 50 percent. There should be more GC content towards the 3′ end so that it’s bound strongly during annealing. Such strong binding is necessary for DNA extension.
- The annealing temperature of the primer can be calculated from equation, 2(AT)+4(GC). The annealing temperatures of the primer pair should be approximately equal.
- Primers with repeating nucleotides should be avoided in order to prevent the binding of such primers to repetitive sequences in the target DNA.
- The primers themselves should not bind with each other in order to prevent dimer formation.
- A primer should not be a palindrome to prevent the formation of a hairpin loop where the 5′ end of the primer binds the 3′ end.
- The melting temperature of the primer should be around 52 to 57 degrees Celcius.
The following websites will help you design a good primer:
- https://www.idtdna.com/
- https://bmcbioinformatics.biomedcentral.com/articles/10.1186/1471-2105-13-134
- https://genome.cshlp.org/content/3/3/S30.full.pdf
- https://eurofinsgenomics.eu/en/ecom/tools/pcr-primer-design/
- https://genome.ucsc.edu/cgi-bin/hgBlat
To know more in detail about PCR, real-time PCR, and its applications, click here.
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