GC Content Percentage
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Primers with a 40-60% GC content ensure stable binding of primer/template. G-C bonds contribute more to the stability (increased melting temperatures) of primer/template binding than do A-T bonds. However, two primer/template complexes with identical GC content can have different melting temperatures because base order influences the overall stability.
It has been reported that GC-rich regions of the target DNA are difficult to amplify, so these regions are generally avoided when choosing a target DNA sequence. This is also true for stretches of polypurines or polypyrimidines, which should be avoided.15
The presence of G or C bases at the 3′ end of primers (GC clamp) helps to promote correct binding at the 3′ end due to the stronger hydrogen bonding of G and C bases.06 However, strings of G and of C can form internal, non-Watson-Crick base pairs that disrupt stable primer binding. Generally, sequences containing more than three repeats of G or of C in sequence should be avoided in the first five bases from the 3′ end of the primer, due to the higher probability ofprimer-dimer formation. A short run of G’s at or near the 5′ end of a primer will not disrupt stable binding because the 5′ positioning does not lead to involvement in disruptive secondary structures. It is best to select primers with a random base distribution.