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General Notes on Primer Design in PCR*

?H [?S+ R ln (c/4)] 273.15C + 16.6 log 10 [K+]

where H is the enthalpy and S is the entropy for helix formation, R is the molar gas constant and c is the concentration of primer. This is most easily accomplished using any of a number of primer design software packages on the market(3). Fortunately, a good working approximation of this value (generally valid for oligos in the 1824 base range) can be calculated using the formula:

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

The table below shows calculated values for primers of various lengths using this equation, which is known as the Wallace formula, and assuming a 50% GC content(4).

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 92C. This parameter will help ensure a more efficient PCR, but is not always necessary for successful PCR. In general, products between 100600 base pairs are efficiently amplified in many PCR reactions. If there is doubt, the product Tm can be calculated using the formula:

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

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

Tm = 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 bein
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