Internal complementary and high GC content can interfere with which performance aspect?

High GC content and internal complementary regions in nucleic acid sequences can significantly interfere with the binding of primers or probes during processes like PCR (Polymerase Chain Reaction) or other amplification techniques. When there is a high percentage of GC base pairs within a target sequence, it leads to increased stability of the DNA duplex due to the stronger hydrogen bonding between G and C compared to A and T. This heightened stability can cause difficulty during the denaturation step, where the two strands must separate for the amplification to occur effectively.

Additionally, internal complementary sequences within the primers or between the primer and target can lead to the formation of secondary structures or hairpins. These structures hinder the primer from effectively binding to the target sequence, which is necessary for the initiation of amplification. If the primers cannot bind to the target due to these issues, the overall efficiency of the amplification process decreases, potentially leading to poor yields or complete failure to amplify the intended sequence.

Therefore, the interference with binding due to high GC content and internal complementarity is a critical performance aspect in molecular diagnostics, particularly in techniques that require precise annealing of primers to target sequences.