Analyze PCR primer properties including melting temperature, GC content, and PCR suitability.
Basic Tm (Wallace rule): 2°C × (A+T) + 4°C × (G+C) — suitable for primers 14–20 nt.
Salt-adjusted Tm: Tm₁ₘ + 16.6 × log₁₀([Na⁺]) — corrects for salt concentration.
Nearest-neighbor Tm (SantaLucia 1998): Most accurate method using thermodynamic ΔH/ΔS parameters for all 16 dinucleotide pairs. Uses CT = 250 nM primer concentration.
Extinction coefficient: A=313.21, T=304.20, G=329.21, C=289.18 g/mol per nucleotide.