For primer Tm = 4(G+C)+2(A+T), Tm of 3 GC and 4 AT is
The Wallace rule approximates melting temperature as Tm = 4(G+C) + 2(A+T), where GC contributes three hydrogen bonds and AT contributes two, providing higher thermal stability for GC pairs. For a primer composition containing three GC and four AT bases, energetic contribution from GC equals 4×3 =12°C and from AT equals 2×4=8°C. Summation yields 20°C, representing temperature at which half of duplex dissociates into single strands. Although modern nearest-neighbor thermodynamic algorithms provide greater accuracy for longer oligos, this formula remains fundamental for NEET-type quick calculations and introductory primer design exercises taught academically.
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.