Hybridization Temperature Estimator

Estimate an approximate nucleic acid melting temperature (Tm) and a simple hybridization temperature range using probe length, GC content, salt concentration, and formamide percentage.

Important: This tool uses simplified formulas for educational and planning purposes. It does not replace validated protocols, manufacturer recommendations, or specialized software. Not for clinical or diagnostic use.

Hybridization Parameters

Probe sequence (optional, 5′ → 3′)

Enter a DNA probe sequence using A, T, G, and C. Non-ATGC characters will be ignored when estimating length and GC content. You can also leave this blank and provide length and GC% manually.

Probe length (nt, optional override)

If you do not provide a sequence, or if you prefer, you can manually enter the probe length in nucleotides.

GC content (% GC, optional override)

If no sequence is provided, you can manually enter the approximate GC percentage (0–100%).

Approximate [Na⁺] (mol/L)

Approximate monovalent cation concentration (e.g., 0.1–1.0 M). Used in a simple long-oligo Tm formula.

Formamide in hybridization buffer (%)

Approximate percentage of formamide in the hybridization mix (e.g., 0–50%). Used for a simple linear Tm reduction.

User-supplied Tm (°C, optional)

If you already have a Tm from another calculator or kit instructions, enter it here and the estimator will use it as the baseline.

Hybrid type

Used only for qualitative interpretation; the numeric formulas remain simple and generic.

Desired stringency (illustrative)

Higher stringency conceptually uses a hybridization temperature closer to Tm; lower stringency uses a lower hybridization temperature. This affects only an illustrative offset and is not a protocol recommendation.

This estimator uses simple rules-of-thumb for Tm and an illustrative offset to suggest a hybridization temperature. It does not replace validated protocols, kits, or primer/probe design tools.

Results

Provide basic probe and buffer information to estimate an approximate Tm and an illustrative hybridization temperature range.

Understanding Hybridization Temperature & Melting Temperature

What is Melting Temperature (Tm)?

The melting temperature (Tm) of a nucleic acid duplex is the temperature at which 50% of the double-stranded molecules have dissociated into single strands. It's a fundamental thermodynamic property that depends on the sequence composition, length, and solution conditions.

Higher GC content leads to higher Tm because G-C base pairs form three hydrogen bonds compared to two for A-T pairs. Longer probes also tend to have higher Tm values due to increased cumulative binding energy.

Tm Calculation Methods

Wallace Rule

Tm = 2×(A+T) + 4×(G+C)

A simple approximation for short oligonucleotides (typically 14-20 bp). Easy to calculate but doesn't account for salt or other factors.

Long-Oligo Formula

Tm = 81.5 + 16.6×log₁₀[Na⁺] + 0.41×(%GC) − 500/length

A simplified formula for longer probes that incorporates salt concentration and length effects. Still an approximation.

Note: More accurate Tm predictions use nearest-neighbor thermodynamic models that consider sequence context, salt corrections, and probe concentration. These simple formulas are for quick estimation only.

What is Hybridization Temperature?

Hybridization temperature is the temperature used during the annealing step of hybridization experiments (e.g., Southern blots, Northern blots, in situ hybridization, microarrays). It's typically set somewhat below the Tm to allow probe-target binding while maintaining specificity.

The relationship between Tm and hybridization temperature is influenced by the desired "stringency" of the experiment.

Understanding Stringency

High Stringency

Hybridization temperature closer to Tm. Favors perfect or near-perfect matches. Reduces non-specific binding but may reduce signal from true targets.

Medium Stringency

A balanced setting for many applications. Allows some mismatches while maintaining reasonable specificity.

Low Stringency

Hybridization temperature well below Tm. Allows more mismatches. Useful for detecting homologous sequences or cross-species hybridization.

Effect of Formamide

Formamide is a denaturant commonly added to hybridization buffers. It lowers the effective Tm, allowing hybridization to occur at lower temperatures. This can reduce secondary structure formation and improve probe access.

A common rule of thumb is that each 1% formamide lowers the Tm by approximately 0.5-0.7°C. This tool uses a simple linear approximation of 0.6°C per 1% formamide.

Factors Not Modeled Here

Divalent cations (Mg²⁺): Stabilize duplexes and affect Tm
Probe/target concentration: Affects kinetics and equilibrium
Secondary structures: Hairpins and self-dimers can compete with hybridization
Mismatches: Single nucleotide differences significantly affect Tm
Surface effects: Immobilized probes (arrays) behave differently than solution
Nearest-neighbor effects: Sequence context matters for accurate Tm

Important Disclaimer

This tool provides simplified estimates for educational and conceptual planning purposes. It does NOT design target-specific probes, provide validated protocol temperatures, or replace proper experimental optimization. Always follow kit instructions, manufacturer recommendations, and your institution's validated protocols for actual experiments. This tool is not intended for clinical or diagnostic applications.

Frequently Asked Questions

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