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GeneFisher 2
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Primer Design
The Polymerase Chain Reaction (PCR) is one of the most important techniques used in life sciences for a variety of tasks. PCR is basically the multiple replication of DNA Fragments and is used for example to amplify known genes, the verification of successful cloning, the identification of organisms and an uncountable number of further applications. The protocol for running a PCR is straightforward. It requires a template DNA, 2 Primer, Polymerase, a mixture of nucleotides (dNTPs), buffer solution and a so called thermocycler is heating and cooling the mixture repeatedly.
The Primers used in the reaction usually are polymers of 20 nucleotides length. Optimal primers are essential for a successful PCR. Primer mark the left and right border of the amplified DNA Fragment, because the 3'-end of the primers is required as starting point for the activity of the Polymerase that creates a copy of the template strand. It is obvious that Primers have to fulfill a variety of different requirements to be effective.
  • In first place the primers have to form a nearly perfect match with the target sequence, especially at their 3'-end.
  • They should contain around 50% GC content. A higher content would increase the primer melting temperature. The melting temperature is the upper border of the annealing temperature of a PCR cycle and a pair of primers should have nearly the same value.
  • Hybridisation of the primer to itself as well as to the other primer has to be avoided by looking at the residue composition.
To avoid the pitfalls that occur when designing primers by hand, some online tools are available that alleviate proper primer selection.