Tm depends on the length of the DNA molecule and its specific nucleotide sequence.
In order to reduce the diversity and obtain the most energetically preferred complexes, a technique called annealing is used in laboratory practice.
However, due to the different molecular geometries of the nucleotides, a single inconsistency between the two strands will make binding between them less energetically favorable.
In the absence of external negative factors, the processes of hybridization and melting may be repeated in succession indefinitely, which lays the ground for polymerase chain reaction.
DNA denaturation, also called DNA melting, is the process by which double-stranded deoxyribonucleic acid unwinds and separates into single-stranded strands through the breaking of hydrophobic stacking attractions between the bases.
Because cytosine / guanine base-pairing is generally stronger than adenine / thymine base-pairing, the amount of cytosine and guanine in a genome is called its GC-content and can be estimated by measuring the temperature at which the genomic DNA melts.
DNA is heated and denatured into single-stranded state, and the mixture is cooled to allow strands to rehybridize.
On a genomic scale, the method has been used by researchers to estimate the genetic distance between two species, a process known as DNA-DNA hybridization.
[4][5] Methods of DNA analysis based on melting temperature have the disadvantage of being proxies for studying the underlying sequence; DNA sequencing is generally considered a more accurate method.
The process of DNA melting is also used in molecular biology techniques, notably in the polymerase chain reaction.
Annealing, in genetics, means for complementary sequences of single-stranded DNA or RNA to pair by hydrogen bonds to form a double-stranded polynucleotide.
The term is also often used to describe the reformation (renaturation) of reverse-complementary strands that were separated by heat (thermally denatured).
DNA strand annealing is a key step in pathways of homologous recombination.
In particular, during meiosis, synthesis-dependent strand annealing is a major pathway of homologous recombination.
For biological systems with water as a solvent, hydrophobic effect contributes and helps in formation of a helix.
[9] Contribution of stacking to the free energy of the molecule can be experimentally estimated by observing the bent-stacked equilibrium in nicked DNA.
According to the Van´t Hoff equation, the relation between free energy, ΔG, and K is ΔG° = -RTln K, where R is the ideal gas law constant, and T is the kelvin temperature of the reaction.
The melting temperature, Tm, occurs when half of the double-stranded nucleic acid has dissociated.
To account for such complicated behavior, the methods of statistical mechanics must be used, which is especially relevant for long sequences.
The previous paragraph shows how melting temperature and thermodynamic parameters (ΔG° or ΔH° & ΔS°) are related to each other.
Vice versa, and important for applications, when the thermodynamic parameters of a given nucleic acid sequence are known, the melting temperature can be predicted.
Instead of treating a DNA helix as a string of interactions between base pairs, the nearest-neighbor model treats a DNA helix as a string of interactions between 'neighboring' base pairs.
In general, the free energy of forming a nucleic acid duplex is
represents the free energy associated with one of the ten possible the nearest-neighbor nucleotide pairs, and
The parameters associated with the ten groups of neighbors shown in table 1 are determined from melting points of short oligonucleotide duplexes.
A more realistic way of modeling the behavior of nucleic acids would seem to be to have parameters that depend on the neighboring groups on both sides of a nucleotide, giving a table with entries like "TCG/AGC".
However, other means exist to access thermodynamic parameters of nucleic acids: microarray technology allows hybridization monitoring of tens of thousands sequences in parallel.
This data, in combination with molecular adsorption theory allows the determination of many thermodynamic parameters in a single experiment[15] and to go beyond the nearest neighbor model.
[16] In general the predictions from the nearest neighbor method agree reasonably well with experimental results, but some unexpected outlying sequences, calling for further insights, do exist.
[16] Finally, we should also mention the increased accuracy provided by single molecule unzipping assays which provide a wealth of new insight into the thermodynamics of DNA hybridization and the validity of the nearest-neighbour model as well.