Concepts
Hybridization
Hybridization is the process of establishing a non-covalent, sequence-specific interaction between two or more complementary strands ofDenaturation
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. See Hydrophobic effect. Both terms are used to refer to the process as it occurs when a mixture is heated, although "denaturation" can also refer to the separation of DNA strands induced by chemicals like formamide or urea. The process of DNA denaturation can be used to analyze some aspects of DNA. Because cytosine / guanine base-pairing is generally stronger than adenine / thymine base-pairing, the amount of cytosine and guanine in a genome (called the " GC content") can be estimated by measuring the temperature at which the genomic DNA melts. Higher temperatures are associated with high GC content. DNA denaturation can also be used to detect sequence differences between two different DNA sequences. DNA is heated and denatured into single-stranded state, and the mixture is cooled to allow strands to rehybridize. Hybrid molecules are formed between similar sequences and any differences between those sequences will result in a disruption of the base-pairing. 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. In the context of a single isolated region of DNA, denaturing gradient gels and temperature gradient gels can be used to detect the presence of small mismatches between two sequences, a process known asAnnealing
Annealing, in genetics, means for complementary sequences of single-stranded DNA or RNA to pair byStacking
Stacking is the stabilizing interaction between the flat surfaces of adjacent bases. Stacking can happen with any face of the base, that is 5'-5', 3'-3', and vice versa. Stacking in "free" nucleic acid molecules is mainly contributed by intermolecular force, specifically electrostatic attraction among aromatic rings, a process also known as pi stacking. For biological systems with water as a solvent, hydrophobic effect contributes and helps in formation of a helix. Stacking is the main stabilizing factor in the DNA double helix. Contribution of stacking to the free energy of the molecule can be experimentally estimated by observing the bent-stacked equilibrium in nicked DNA. Such stabilization is dependent on the sequence. The extent of the stabilization varies with salt concentrations and temperature.Thermodynamics of the two-state model
Several formulas are used to calculate ''Tm'' values. Some formulas are more accurate in predicting melting temperatures of DNA duplexes. For DNA oligonucleotides, i.e. short sequences of DNA, the thermodynamics of hybridization can be accurately described as a two-state process. In this approximation one neglects the possibility of intermediate partial binding states in the formation of a double strand state from two single stranded oligonucleotides. Under this assumption one can elegantly describe the thermodynamic parameters for forming double-stranded nucleic acid AB from single-stranded nucleic acids A and B. :AB ↔ A + B The equilibrium constant for this reaction is . According to the Van´t Hoff equation, the relation between free energy, Δ''G'', and ''K'' is Δ''G°'' = -''RT''ln ''K'', where ''R'' is the ideal gas law constant, and ''T'' is the kelvin temperature of the reaction. This gives, for the nucleic acid system, . The melting temperature, ''T''m, occurs when half of the double-stranded nucleic acid has dissociated. If no additional nucleic acids are present, then and Bwill be equal, and equal to half the initial concentration of double-stranded nucleic acid, Bsub>initial. This gives an expression for the melting point of a nucleic acid duplex of . Because Δ''G''° = Δ''H''° -''T''Δ''S''°, ''T''m is also given by . The terms Δ''H''° and Δ''S''° are usually given for the association and not the dissociation reaction (see the nearest-neighbor method for example). This formula then turns into: , where sub>total ≤ sub>total. As mentioned, this equation is based on the assumption that only two states are involved in melting: the double stranded state and the random-coil state. However, nucleic acids may melt via several intermediate states. To account for such complicated behavior, the methods ofEstimating thermodynamic properties from nucleic acid sequence
The previous paragraph shows how melting temperature and thermodynamic parameters (Δ''G''° or Δ''H''° & Δ''S''°) are related to each other. From the observation of melting temperatures one can experimentally determine the thermodynamic parameters. Vice versa, and important for applications, when the thermodynamic parameters of a given nucleic acid sequence are known, the melting temperature can be predicted. It turns out that for oligonucleotides, these parameters can be well approximated by the nearest-neighbor model.Nearest-neighbor method
The interaction between bases on different strands depends somewhat on the neighboring bases. 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. So, for example, the DNA shown below has nearest-neighbor interactions indicated by the arrows. : ↓ ↓ ↓ ↓ ↓
:5' C-G-T-T-G-A 3'
:3' G-C-A-A-C-T 5'
The free energy of forming this DNA from the individual strands, Δ''G''°, is represented (at 37 °C) as
Δ''G''°37(predicted) = Δ''G''°37(C/G initiation) + Δ''G''°37(CG/GC) + Δ''G''°37(GT/CA) + Δ''G''°37(TT/AA) + Δ''G''°37(TG/AC) + Δ''G''°37(GA/CT) + Δ''G''°37(A/T initiation)
Except for the C/G initiation term, the first term represents the free energy of the first base pair, CG, in the absence of a nearest neighbor. The second term includes both the free energy of formation of the second base pair, GC, and stacking interaction between this base pair and the previous base pair. The remaining terms are similarly defined. In general, the free energy of forming a nucleic acid duplex is
,
where represents the free energy associated with one of the ten possible the nearest-neighbor nucleotide pairs, and represents its count in the sequence.
Each Δ''G''° term has enthalpic, Δ''H''°, and entropic, Δ''S''°, parameters, so the change in free energy is also given by
.
Values of Δ''H''° and Δ''S''° have been determined for the ten possible pairs of interactions. These are given in Table 1, along with the value of Δ''G''° calculated at 37 °C. Using these values, the value of Δ''G''37° for the DNA duplex shown above is calculated to be −22.4 kJ/mol. The experimental value is −21.8 kJ/mol.
The parameters associated with the ten groups of neighbors shown in table 1 are determined from melting points of short oligonucleotide duplexes. Curiously, it works out that only eight of the ten groups are independent.
The nearest-neighbor model can be extended beyond the Watson-Crick pairs to include parameters for interactions between mismatches and neighboring base pairs. This allows the estimation of the thermodynamic parameters of sequences containing isolated mismatches, like e.g. (arrows indicating mismatch)
: ↓↓↓
:5' G-G-A-C-T-G-A-C-G 3'
:3' C-C-T-G-G-C-T-G-C 5'
These parameters have been fitted from melting experiments and an extension of Table 1 which includes mismatches can be found in literature.
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, this would involve around 32 groups for Watson-Crick pairing and even more for sequences containing mismatches; the number of DNA melting experiments needed to get reliable data for so many groups would be inconveniently high. 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 and to go beyond the nearest neighbor model. 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. 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.
See also
* Melting point *References
External links