PCR is a technology born of the modern molecular biology era. Two critical PCR components are left to the researcher. The first is the nucleic acid template, which should be of sufficient quality and contain no inhibitors of Taq DNA polymerase. The second is the selection of the oligonucleotide primers. This process is often critical for the overall success of a PCR experiment, for without a functional primer set, there will be no PCR product. Efficacy and sensitivity of PCR largely depend on the efficiency of primers. The ability for an oligonucleotide to serve as a primer for PCR is dependent on several factors including:

a) The kinetics of association and dissociation of primer-template duplexes at the annealing and extension temperatures;

b) The efficiency with which the polymerase can recognize and extend a mismatched duplex

The primers which are unique for the target sequence to be amplified should fulfill certain criteria such as primer length, GC%, annealing and melting temperature, 5' end stability, 3' end specificity etc. Maybe the most critical parameter for successful PCR is the design of Primers. A badly designed primer can result in little or no product due to non-specific amplification and/or primer-dimer formation, which can become competitive enough to suppress product formation. When choosing two PCR amplification primers, the following guidelines should be considered:

Primer length: Primers of typically 18-30 nucleotides in length are the best. Primers should be at least 18 nucleotides in length to minimise the chances of encountering problems with a secondary hybridisation site on the vector or insert. Primers with long runs of a single base should generally be avoided. It is especially important to avoid 4 or more G's or C's in a row.

Melting Temperature (Tm): The optimal melting temperatures for primers in the range 52-58^oC, generally produce better results than primers with lower melting temperatures. Primers with melting temperatures above 65oC should also be avoided because of potential for secondary annealing. A good working approximation of this value (generally valid for oligos in the 18–30 base range) can be calculated using the formula of Wallace et al. (1979),

Tm= 2(A+T) + 4(G+C).

Two issues are critical for Tm:

1. The two primers should have a similar Tm

2. The Tm should be within 55-72ºC, around 60ºC is ideal.

GC Content: GC% is an important characteristic of DNA and provides information about the strength of annealing. Primers should have GC content between 45 and 60 percent. For primers with a G/C content of less than 50%, it may be necessary to extend the primer sequence beyond 18 bases to keep the melting temperature above the recommended lower limit of 50oC. GC content, melting temperature and annealing temperature are strictly dependent on one another.

3’-End Sequence: It is well established that the 3' terminal position in PCR primers is essential for the control of mis-priming. Primers should be "stickier" on their 5' ends than on their 3' ends. A "sticky" 3' end as indicated by a high G/C content could potentially anneal at multiple sites on the template DNA. A "G" or "C" is desirable at the 3' end but the first part of this rule should apply.

Dimers and false priming cause misleading results: Primers should not contain complementary (palindromes) within themselves; that is, they should not form hairpins. If this state exists, a primer will fold back on itself and result in an unproductive priming event that decreases the overall signal obtained. Primers should not contain sequences of nucleotides that would allow one primer molecule to anneal to itself or to the other primer used in PCR reactions (primer dimer formation).

Specificity: As mentioned above, primer specificity is at least partly dependent on primer length. It is evident that there are many more unique 24 base oligos than there are 15 base pair oligos. However, primers must be chosen so that they have a unique sequence within the template DNA that is to be amplified. A primer designed with a highly repetitive sequence will result in a smear when amplifying genomic DNA. However, the same primer may give a single band if a single clone from a genomic library is amplified.

Complementary primer sequences: Primers need to be designed with absolutely no intra-primer homology beyond 3 base pairs. If a primer has such a region of self-homology, "snap back" can occur. Another related danger is inter-primer homology: partial homology in the middle regions of two primers can interfere with hybridization. If the homology should occur at the 3' end of either primer, primer dimer formation will occur.