SSO probes should be designed to have their target mismatches where to maximize specificity?

The design of SSO (sequence-specific oligonucleotide) probes necessitates careful consideration of where mismatches are placed to optimize specificity in hybridization. When mismatches are located in the center of the probe, they significantly diminish the ability of the probe to hybridize with its target sequence accurately. This is because the central nucleotides play a crucial role in stabilizing the overall affinity of the probe for the target—any mismatch in this region can dramatically decrease binding stability, thereby enhancing specificity.

The ends of the probe also contribute to binding, but they are generally more tolerant of mismatches, which can sometimes still allow for hybridization. Therefore, central mismatches would typically result in a greater chance of rejecting non-matching sequences, thus improving specificity. The correct placement of mismatches at the center is a common strategy in probe design to ensure that only sequences with perfect complementarity are detected, leading to more reliable results in applications such as genotyping and molecular diagnostics.