Primer (molecular Biology) - PCR Primer Design

PCR Primer Design

Pairs of primers should have similar melting temperatures since annealing in a PCR occurs for both simultaneously. A primer with a T_m significantly higher than the reaction's annealing temperature may mishybridize and extend at an incorrect location along the DNA sequence, while T_m significantly lower than the annealing temperature may fail to anneal and extend at all.

Primer sequences need to be chosen to uniquely select for a region of DNA, avoiding the possibility of mishybridization to a similar sequence nearby. A commonly used method is BLAST search whereby all the possible regions to which a primer may bind can be seen. Both the nucleotide sequence as well as the primer itself can be BLAST searched. The free NCBI tool Primer-BLAST integrates primer design tool and BLAST search into one application, so does commercial software product such as ePrime, Beacon Designer. Computer simulations of theoretical PCR results (Electronic PCR) may be performed to assist in primer design. Mononucleotide repeats should be avoided, as loop formation can occur and contribute to mishybridization. Primers should not easily anneal with other primers in the mixture (either other copies of same or the reverse direction primer); this phenomenon can lead to the production of 'primer dimer' products contaminating the mixture. Primers should also not anneal strongly to themselves, as internal hairpins and loops could hinder the annealing with the template DNA.

When designing a primer for use in TA cloning, efficiency can be increased by adding AG tails to the 5' and the 3' end.

The reverse primer has to be the reverse complement of the given cDNA sequence. The reverse complement can be easily determined, e.g. with on-line calculators.