...
The polymerase chain reaction (PCR) uses a pair of custom primers to direct DNA elongation toward each other at opposite ends of the sequence being amplified. These primers are typically between 18 and 24 bases in length and must code for only the specific upstream and downstream sites of the sequence being amplified. A primer that can bind to multiple regions along the DNA will amplify them all, eliminating the purpose of PCR.
A few criteria must be brought into consideration when designing a pair of PCR primers. Pairs of primers should have similar melting temperatures since annealing during PCR occurs for both strands simultaneously, and this shared melting temperature must not be either too much higher or lower than the reaction's annealing temperature. A primer with a Tm (melting temperature) too much higher than the reaction's annealing temperature may mishybridize and extend at an incorrect location along the DNA sequence. A Tm significantly lower than the annealing temperature may fail to anneal and extend at all.
Additionally, primer sequences need to be chosen to uniquely select for a region of DNA, avoiding the possibility of hybridization to a similar sequence nearby.
Also, there are other parameters to be taken in account - Gibbs free energy, self- and heterodimers availability, etc. All parameters are described below in details.
In silico PCR is used to calculate theoretical polymerase chain reaction (PCR) results using a given set of primers (probes) to amplify DNA sequences.
UGENE provides the In silico PCR "PCR Primer Design for DNA Assembly" feature only for nucleic sequences with the "Standard DNA and Extended DNA alphabets" alphabet. To use it in UGENE open a DNA sequence and go to the In silico PCR"PCR Primer Design for DNA Assembly" tab of the Options Panel:
...