GC Content & Tm Calculator

GC content is the percentage of Guanine (G) and Cytosine (C) bases in a DNA or RNA sequence. It is a key indicator of the thermal stability of the nucleic acid.

It directly affects the melting temperature (Tm) of a primer. Primers with GC content between 40-60% generally provide a good balance of stability for specific binding without being so stable that they are difficult to melt during the denaturation step of PCR.

Tm is the temperature at which 50% of double-stranded DNA molecules have denatured (separated) into single strands. It is a critical parameter for setting the annealing temperature in PCR.

The annealing temperature (Ta) is the step where primers bind to the template DNA. It is typically set about 3-5°C below the lowest Tm of the primer pair to ensure specific and efficient binding.

Guanine and Cytosine form three hydrogen bonds between them, while Adenine and Thymine form only two. The extra hydrogen bond in a G-C pair makes it thermodynamically more stable and requires more energy (heat) to break apart.

The Wallace Rule is a very basic formula for estimating Tm, often used for short primers (<20 bases). It is calculated as Tm = 2°C × (A+T) + 4°C × (G+C). This tool uses this basic method.

Yes. More advanced formulas, like the Salt-Adjusted or nearest-neighbor thermodynamic calculations, are much more accurate because they account for factors like salt concentration (Na⁺), primer concentration, and other thermodynamic parameters.

Positive ions (like Na⁺) in a solution shield the negative charges on the phosphate backbones of the DNA strands. This reduces the electrostatic repulsion between the strands, making the double helix more stable and thus increasing its melting temperature.

A GC clamp refers to having one or two G or C bases at the 3' end of a primer. Because the 3' end is where DNA polymerase begins synthesis, this stronger binding helps promote efficient and specific amplification. However, more than two G/C bases should be avoided to prevent non-specific binding.

Primers are typically between 18 and 25 bases long. This length is generally sufficient to ensure the sequence is unique in the target genome, preventing off-target binding.

For GC content calculation, this tool will ignore any ambiguous bases (like 'N') as they are neither G nor C. For the basic Tm calculation, ambiguous codes are not factored in.

No. This is a simple calculator for GC content and basic Tm. Checking for secondary structures like hairpins (where a primer binds to itself) or primer-dimers (where primers bind to each other) requires specialized bioinformatics software.

This calculator is specifically designed for DNA. It will count 'G' and 'C' in an RNA sequence for GC content, but the Tm formula is not valid for RNA.

Primers with very low GC content (<40%) may have a Tm that is too low, leading to weak or no binding. Primers with very high GC content (>60%) can have a high Tm and be prone to non-specific binding and the formation of secondary structures.

Ideally, yes. For optimal PCR performance, the Tm of the forward and reverse primers should be within 5°C of each other. This ensures that both primers bind to the template with similar efficiency at the chosen annealing temperature.

You can paste raw sequence data, even with spaces or numbers. The tool will automatically strip any characters that are not A, T, G, or C before performing the calculation.

No. GC content can vary significantly across different regions of a genome. GC-rich regions are often associated with gene-coding areas, while AT-rich regions are common in non-coding or structural parts of the chromosome.

GC skew is a measure of whether Gs or Cs are more abundant on a particular strand of DNA. It's an advanced metric used in genomics, for example, to help identify the origin of replication.

Yes, GC content and Tm are equally critical for designing probes used in quantitative PCR (qPCR) or Fluorescence In Situ Hybridization (FISH). Probes often have higher Tm values than primers.

No. While it provides a basic check, designing primers for clinical or diagnostic applications is a highly regulated process that requires certified software and extensive validation to ensure specificity and reliability.

Primer3 (often available via web interfaces) is a widely used and powerful tool for primer design. The NCBI also offers Primer-BLAST, which designs primers and then checks them for specificity against a sequence database.

PCR can fail for many reasons besides incorrect annealing temperature, such as poor template quality, presence of inhibitors, incorrect magnesium concentration, or issues with the primers themselves (e.g., secondary structures).

Use a calculator that employs the salt-adjusted or nearest-neighbor thermodynamic models and allows you to input the concentrations of salt (Na+), Mg2+, and dNTPs in your PCR mix.

The basic Tm formulas are only valid for short oligonucleotides (<100 bp). For long DNA molecules like plasmids or chromosomes, the melting behavior is more complex and cannot be represented by a single Tm value using these simple formulas.

Yes. All calculations are performed entirely in your browser. Your sequence data is never sent to our servers, ensuring your research remains confidential.