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GC Content Percentage

Home > DNA Amplification > Overview > Primer Design > GC Content Percentage

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 of primer-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.

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NOTE TO USERS: The President’s DNA Initiative DNA Analyst Training program and assessment were completed and published in 2005, in cooperation with the National Institute of Justice. The science and techniques in the program are sound and proven, however, program content has not been updated to include tools and technologies developed and in use after 2005, including many kits and robots. Assessment questions address only content delivered in this program and may not contain the full range of tools in use in your laboratory.