( re on the annealing temperature in PCR ...)Optimizing DNA Amplification Protocols using the Eppendorf Mastercycler

The sequence plays a very important part. The ideal G/C content generally lies in the range of 4555%. In order to insure stable annealing, the primer should, as far as possible, be complementary to the desired DNA sequence and, in addition, be able to form G/C clamps, i.e. several consecutive G/C or C/G base pairs, between the 3' end of the primer and the template DNA (5).

Self-complementarity within a primer by virtue of palindromes or long segments of polypurines and polypyrimides, or areas complementary to the sequence of the second primer, enhance the formation of primer dimers and should therefore be avoided.

Selection of primer sequences and the calculation of annealing temperatures (see below) can be computer-aided using a range of software.

Both primers should have similar Tm values, which can be most easily calculated according to the Wallace formula [(A/T)x2 plus (G/C)x4] (6). This simple rule, however, is restricted to primers with a maximum length of 20 nucleotides. Longer primers require more complex formulas (7).

Ideally, the Tm values should lie in the range of 5565C. PCR optimization often begins at an annealing temperature of 5C below the primers' Tm.

The optimum annealing temperature, however, often enough is considerably higher than the calculated Tm. One optimization strategy is therefore to run additional PCRs, gradually increasing the annealing temperature each time by 25C.

The so-called touch down PCR is
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