PCR Primer Design and Bioinformatics

The specificity of the PCR lies in the design of the two oligonucleotide primers. These not only have to be complementary to sequences flanking the target DNA but also must not be self-complementary or bind each other to form dimers, since both prevent authentic DNA amplification. They also have to be matched in their GC content and have similar annealing temperatures and be incapable of amplifying unwanted genomic sequences. Manual design of primers is time consuming and often hit or miss, although equations such as the following are still used to derive the annealing temperature (Ta) for each primer:
                 4(G + C) + 2(A + T)  =  Tm
where Tm is the melting temperature of the primer/target duplex and G, C, A and T are the numbers of the respective bases in the primer. In general, Ta is set 3–5 1C lower than Tm. On occasions, secondary or primer dimer bands may be observed on the electrophoresis gel in addition to the authentic PCR product. In such situations, Touchdown or Hot start regimes may help. Alternatively, raising Ta closer to Tm can enhance the specificity of the reaction.
The increasing use of bioinformatics resources such as Oligo, Generunner and Primer Design Assistant16 in the design of primers makes the design and the selection of reaction conditions much more straightforward. These computer-based resources allow the sequences to be amplified, primer length, product size, GC content, etc., to be input and following analysis, provide a choice of matched primer sequences. Indeed, the initial selection and design of primers without the aid of bioinformatics would now be unnecessarily time consuming. Finally, before ordering or synthesising the primers, it is wise to submit proposed sequences to a nucleotide sequence search program such as BLAST, which can be used to interrogate GenBank or other comprehensive public DNA sequence databases to increase confidence that the reaction will be specific for the intended target sequence.