Biological filtering ‑ GC content

While G-C bonds contribute more to the stability, and therefore increased melting temperature, of primer/template binding than do A−T bonds, it is important to note that two primer/template complexes with similar or even identical GC content can result in a different melting temperature because of base order influence on overall stability.

GC-rich regions for the target DNA are difficult to amplify, and are generally avoided when defining an in silico algorithm.