Ideal GC content for primer is
Primer composition influences melting temperature, specificity, and secondary structure. GC content of 40 to 60 percent ensures balanced stability with melting temperature 55-65°C, optimal for specific annealing at typical PCR conditions. Lower GC content below 30 percent yields weak binding, low Tm, and unstable duplexes, whereas exceeding 70 percent promotes extremely stable secondary structures, hairpins, and non-specific annealing to GC-rich genomic regions, raising mispriming risk. Balanced GC content supports efficient denaturation and specific hybridization, as taught in NEET, CUET, and
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.