Tm of a primer is calculated by
Melting temperature of short oligonucleotides dictates annealing conditions and assay success. The Wallace rule provides rapid estimation for primer design: Tm = 4°C × (G+C) + 2°C × (A+T). Guanine-cytosine pairs form three hydrogen bonds contributing greater thermal stability than adenine-thymine pairs with two bonds, thus weighted higher in formula. This approximation applies to primers shorter than about 20 bases and guides optimization of annealing temperature in PCR and hybridization experiments. Accurate Tm prediction ensures specific primer binding, minimizing non-specific products while
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.