The formula for calculating melting temperature (Tm) is:
Melting temperature represents the temperature at which fifty percent of an oligonucleotide duplex dissociates. For short oligonucleotides, typically less than 14 nucleotides, Wallace rule provides a rapid empirical estimate. Guanine-cytosine pairs form three hydrogen bonds and exhibit stronger stacking, contributing roughly 4 degrees Celsius per pair, while adenine-thymine pairs form two hydrogen bonds, contributing about 2 degrees per pair. Summation yields Tm = 4(G+C) + 2(A+T). This approximation guides primer design for PCR and probe hybridization, though more accurate nearest-neighbor the
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.