A primer is a segment of nucleic acid that serves as a starting point for DNA synthesis.
It is essential for artificial replication because DNA polymerase cannot construct a
strand of DNA from scratch and can instead only add nucleotides to the 3'-end of an existing strand.
Primer design is a key component in any PCR experiment, and there is some common guidelines to the good primers:
Primer Length. 18-22 is optimal, it is long enough for adequate specificity and short enough for primers to bind
easily to the template at the annealing temperature.
Primer Melting Temperature (Tm). It is a temperature at which double-strand DNA (in this case duplex
"primer-target region") will dissociate. Tm depends on the GC-content of the primer (because GC-pairs is more
stable than AT, and need more energy to separate). Best results generally produced in the range of 52-58 °C
Primer annealing temperature (Ta, second step of the PCR cycle). Typically is about 3-5 °C below the Tm of the
primers used. Higher annealing temperature means higher specificity, but resulting in lower PCR product yield.
Lower Ta may possibly lead to non-specific products because of a high number of base pair mismatches.
