The temperatures calculated using Wallace's rule are inaccurate at the extremes of this chart.

In addition to calculating the melting temperatures of the primers, care must be taken to ensure that the melting temperature of the product is low enough to ensure 100% melting at 92 °C. This parameter will help ensure a more efficient PCR, but is not always necessary for successful PCR. In general, products between 100–600 base pairs are efficiently amplified in many PCR reactions. If there is doubt, the product T_m can be calculated using the formula:

T_m = 81.5 + 16.6 (log₁₀[K+] + 0.41 (%G+C)–675/length.

Under standard PCR conditions of 50 mM KCL, this reduces to⁽³⁾:

T_m = 59.9 + 0.41 (%G+C) – 675/length

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. That being said, 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.

Because Taq Polymerase is active over a broad range of temperatures, primer extension will occur at the lower temperatures of annealing. If the temperature is too low, non-specific priming may occur which can be extended by the polymerase if there is a short homology at the 3' end. In general, a melting temperature of 55 °C –72 °C gives the best results (Note that this corresponds to a primer length of 18–24 bases using Wallace's rule above).

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”, partially double-stranded structures, can occur which will interfere with annealing to the template.

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 which, more often than not, will prevent the formation of the desired product via competition.

G/C content and Polypyrimidine (T, C) or polypurine (A, G) stretches

The base composition of primers should be between 45% and 55% GC. The primer sequence must be chosen such that there is no PolyG or PolyC stretches that can promote non-specific annealing. Poly A and Poly T stretches are also to be avoided as these will “breath” and open up stretches of the primer-template complex. This can lower the efficiency of amplification. Polypyrimidine (T, C) and polypurine (A, G) stretches should also be avoided. Ideally the primer will have a near random mix of nucleotides, a 50% GC content and be ~20 bases long. This will put the T_m in the range of 56 °C – 62 °C⁽¹⁾.

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