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General Notes on Primer Design in PCR*

g 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 55C 72C gives the best results (Note that this corresponds to a primer length of 1824 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 tha
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