Primer Melting Temperature (Tm)

Nearest-Neighbor model (SantaLucia 1998) with Owczarzy salt correction · T_m = ΔH / (ΔS + R·ln(C_T/4))

Sequence

Examples:

Primer sequence (5′ → 3′)

Accepts A, C, G, T and IUPAC degenerate codes (R, Y, N…); anything else is ignored.

Length: 20 nt · G+C: 11

Reaction conditions

Parameter set

Oligo concentration

Total strand concentration (C_T).

12000

Sodium / monovalent

Na⁺ and K⁺ from the buffer.

01000

Magnesium

Total Mg²⁺ in the reaction.

010

dNTPs

Chelate Mg²⁺ in a 1:1 ratio.

Results

Melting temperature (Tm)

59.67

°C

Suggested annealing

54.67

°C

Rule of thumb: Ta ≈ Tm − 5 °C.

Thermodynamics

ΔG°₃₇: -25.61 kcal/mol
ΔH: -160.4 kcal/mol
ΔS: -434.6 cal/(K·mol)
3′ end ΔG: -5.069 kcal/mol

Composition

G+C content: 55 %
Length: 20 nt
Self-complementary: No

Melting curve

Fraction of denatured strand vs. temperature (idealized two-state model)

Fundamentals & Explanation

What is the Tm and why does it matter?

The melting temperature (Tm) is the temperature at which half of a DNA duplex is base-paired and the other half has separated into single strands. In a PCR, the primers must bind their template during the annealing step: if the temperature is too high relative to the Tm, the primer won't anchor; if it's too low, it binds at incorrect sites (mispriming). Knowing each primer's Tm is therefore the first step toward a specific, efficient reaction.

The annealing temperature programmed into the thermocycler is usually set a few degrees below the Tm. A common rule of thumb is:

T_a \approx T_m - 5\,°C

The Nearest-Neighbor model

The stability of a duplex depends not only on how many G·C and A·T pairs it has, but on how neighboring bases stack. The Nearest-Neighbor model assigns enthalpy (ΔH) and entropy (ΔS) values to each of the 10 unique dinucleotide steps (AA/TT, AT, TA, CA/TG, …) and sums them along the sequence, adding initiation penalties at each end:

\Delta H = \sum_i \Delta H_{neighbor,i} + \Delta H_{init}

\Delta S = \sum_i \Delta S_{neighbor,i} + \Delta S_{init}

With the total enthalpy and entropy, the Tm of a non-self-complementary oligo follows from the thermodynamics of the two-state transition:

T_m = \dfrac{\Delta H}{\Delta S + R\,\ln\!\left(\dfrac{C_T}{4}\right)}

where R is the gas constant (1.987 cal·K⁻¹·mol⁻¹) and C_T the total strand concentration. The factor of 4 applies to non-self-complementary strands at equal concentration; if the sequence is a palindrome (self-complementary), 1 is used and a symmetry correction is added to the entropy. (The output of this equation is in Kelvin; the calculator displays it converted to °C.)

Reference: SantaLucia (1998), PNAS — unified Nearest-Neighbor parameters

The melting curve

If we track the fraction of denatured strand as the temperature rises, we get a sigmoidal curve whose midpoint (fraction = 0.5) is exactly the Tm. Under the two-state model, that fraction is described by a van 't Hoff-type equation (with temperatures T and Tm in Kelvin):

f(T) = \dfrac{1}{1 + \exp\!\left[\dfrac{\Delta H}{R}\left(\dfrac{1}{T_m} - \dfrac{1}{T}\right)\right]}

The curve drawn here is idealized: it treats the transition as if it were unimolecular to clearly show the slope and the midpoint at the Tm. Real primer–template hybridization is bimolecular and its exact shape depends on concentration, but the midpoint still coincides with the calculated Tm.

The effect of salts

The Nearest-Neighbor parameters are calibrated at 1 M NaCl, but a PCR runs with far less salt. Cations screen the repulsion between the negatively charged phosphate backbones: more salt means a more stable duplex and a higher Tm. This calculator corrects the Tm with the Owczarzy model, which distinguishes the contribution of monovalent cations (Na⁺, K⁺) from divalent Mg²⁺, selecting the dominant regime from the ratio R = √[Mg²⁺] / [monovalent].

A key PCR detail: dNTPs chelate Mg²⁺ in a 1:1 ratio, so what actually stabilizes the duplex is the free magnesium:

[Mg^{2+}]_{free} = [Mg^{2+}]_{total} - [dNTP]

References: Owczarzy et al. (2004), Biochemistry · Owczarzy et al. (2008), Biochemistry

Designing a good primer

Beyond the Tm, the calculator checks several practical design criteria:

•Length of 18–24 nt: long enough to be specific without inflating synthesis cost.
•G+C content of 40–60 %: balances stability and specificity.
•GC clamp: 1–2 G/C bases at the 3′ end help anchoring; too many increase mispriming.
•3′ end stability (ΔG of the last nucleotides): if too negative, the primer may extend at incorrect sites.
•Avoid runs of ≥ 4 identical bases, which cause polymerase slippage.

Degenerate bases (IUPAC). Many real primers —in virology or for the 16S gene— include ambiguous positions (R = A/G, Y = C/T, N = any…) to cover template variants. The calculator expands each code into all of its concrete combinations and reports the Tm (and ΔG, ΔH, ΔS, %GC) as a range between the weakest and the most stable variant. In that case the Ta is computed from the minimum Tm: a higher one would leave the least-stable variants unannealed and lost from the pool.

This tool estimates the thermodynamic properties of a single strand; it does not analyze primer hairpins or dimers (secondary structures), which require a separate folding calculation.

Primer Melting Temperature (Tm)

Sequence

Reaction conditions

Results

Thermodynamics

Composition

Melting curve

Fundamentals & Explanation

What is the Tm and why does it matter?

The Nearest-Neighbor model

The melting curve

The effect of salts

Designing a good primer

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What is the Tm and why does it matter?

The Nearest-Neighbor model

The melting curve

The effect of salts

Designing a good primer