Primer hybridization
As primer hybridization and primer annealing refers to the process of attachment of a primer to DNA sequences, usually in connection with a polymerase chain reaction (PCR).
If the DNA strands are single-stranded after denaturation has taken place , the likewise single-stranded primers are able to bind complementarily to the corresponding target DNA sequence. The annealing temperature describes the maximum temperature at which the polynucleotide sequence still binds to the complementary DNA strand. The theoretical annealing temperature results from the calculation of the free enthalpies of the bond strengths and the practical annealing temperature results from the determination by melting curve analyzes .
The selected annealing temperature is decisive for the specificity of the binding to the target sequence during a PCR. If the temperature is too low, “loose” unspecific bindings of the primers can result and, under certain circumstances, undesirable products. If the temperature is too high, no product is created. As a rule it can be formulated “The higher the temperature, the more specific the annealing to the target sequence”.
This can be imagined as follows: At a low temperature, the base pairings do not always have to match 100%, which can result in loose bonds. If the temperature is increased, such compounds melt because their binding energy is not large enough. A maximum match of the base pairings results in a maximum binding strength and thus also an optimal specificity.
However, this only works until the melting temperature of the primer and target sequence is reached. Therefore, with standard PCRs, a temperature is usually chosen that is about 3 ° C below the calculated melting temperature, so that annealing of the primers to the target sequence is ensured.
See also
literature
- Friedrich Lottspeich, Joachim W. Engels (Ed.): Bioanalytik . 3. Edition. Springer Spectrum, Berlin Heidelberg 2012, ISBN 978-3-8274-2942-1 .