What can be a consequence of using a high GC content in probes?

Using high GC content in probes can lead to interference with binding performance due to several reasons related to the properties of nucleic acid interactions. Probes with high GC content tend to form more stable duplexes compared to those with lower GC content. While this stability is beneficial in certain contexts, it can also create challenges in hybridization efficiency.

When probes have a high GC content, the melting temperature (Tm) increases, which means that they require higher temperatures to dissociate or hybridize effectively. If the hybridization conditions are not optimized to match this increased stability, the probes may not bind as effectively to their target, leading to potential issues in the specificity and sensitivity of the assay.

Moreover, the rigidity of GC-rich regions can lead to difficulties in the bending and flexibility required during the hybridization process. This can result in less effective binding to targets that may have mismatches or variations, ultimately interfering with overall performance. Therefore, while high GC content can enhance binding stability in some cases, it can also complicate the hybridization dynamics, adversely affecting binding performance.