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GC contentMelting temperaturePrimerReverse complementTemplate

GC content

The GC content of a primer is the percentage of nucleotides that are either a G or C nucleotides. G-C base pairs have 3 hydrogen bonds rather than just 2 like A-T base pairs. Thus, in comparing two primers with equal length, the one with the higher GC content will have a higher melting temperature.

Melting temperature

The melting temperature of a primer, abbreviate Tm is defined as the temperature at which 50% of that same DNA molecule species form a stable double helix and the other 50% have been separated to single strand molecules. The melting temperature depends on both primer length and sequence. A good rule of thumb for calculating melting temperatures is 4°C*(# G/C nucleotides) + 2°C*(# A/T nucleotides). [This is the rule used to calculate melting temperature in the primer catalog tables. However, to more accurately calculate melting temperature, you can also use one of several online tools. For PCR and sequencing applications, primers should have a melting temperature of 55-65°C, which generally corresponds to a primer 20-25 nucleotides in length with about 40% GC content.

The melting temperature can also be known as the annealing temperature in reference to the temperature at which primers start to bind template DNA during PCR.


A short, synthetic, single-stranded DNA sequence that binds to a target DNA sequence and enables addition of new deoxyribonucleotides by DNA polymerase at the 3' end.

Primers are also often called oligonucleotides or oligos. In the context of PCR and DNA sequencing, the term "primer" is generally used, in reference to the idea that the synthetic DNA "primes" where copying should begin. The term "oligonucleotide" or "oligo" is more commonly used in the context of ordering synthetic DNA fragments or in the context of DNA synthesis in which synthetic DNA fragments are assembled to make larger pieces of DNA.

Reverse complement

The reverse complement of a DNA sequence is the sequence "backwards" and with each based changed to its pair (G to C, C to G, A to T and T to A). For example, the DNA sequence ATGC becomes GCAT. To obtain the reverse complement of a DNA sequence, you can use one of several online tools like this one.


The target DNA sequence to which a primer is supposed to anneal.