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#GC content

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PCR

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 calculatio

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.

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PCR

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.

View Details