The stringency of hybridization is highest when:
Stringency defines the permissiveness of hybridization conditions. High stringency allows only perfectly complementary duplexes to remain annealed, while mismatched hybrids dissociate. Elevated temperature increases thermal energy, melting imperfect duplexes due to lower thermodynamic stability. Low salt reduces cation concentration, minimizing shielding of phosphate repulsion, thereby raising electrostatic barrier to annealing and destabilizing weak interactions. Conversely, low temperature and high salt represent low stringency, favoring annealing of partially mismatched sequences. Molecular
Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.