Nucleic acid thermodynamics is the study of the thermodynamics of nucleic acid molecules, or how temperature affects nucleic acid structure. For multiple copies of DNA molecules, the melting temperature (Tm) is defined as the temperature at which half of the DNA strands are in the double-helical state and half are in the "random-coil" states.[1] The melting temperature depends on both the length of the molecule, and the specific nucleotide sequence composition of that molecule.
Hybridization Hybridization is the process of establishing a non-covalent, sequence-specific interaction between two or more complementary strands of nucleic acids into a single hybrid, which in the case of two strands is referred to as a duplex. Oligonucleotides, DNA, or RNA will bind to their complement under normal conditions, so two perfectly complementary strands will bind to each other readily. In order to reduce the diversity and obtain the most energetically preferred hybrids, a technique called annealing is used in the 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. Measuring the effects of base incompatibility by quantifying the rate at which two strands anneal can provide information as to the similarity in base sequence between the two strands being annealed. The hybrids may be dissociated by thermal denaturation also referred to as melting. Here, the solution of hybrids is heated to break the hydrogen bonds between nucleic bases, after which the two strands separate. In the absence of external negative factors, the processes of hybridization and melting may be repeated in succession indefinitely long, which lays the ground for polymerase chain reaction. Most commonly, the pairs of nucleic bases A=T and G≡C are formed, of which the latter is more stable. Denaturation 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 hydrogen bonding between the bases. 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 urea[citation needed] . The process of DNA denaturation can be used to analyze some aspects of DNA. Because cytosine / guanine base-pairing is generally stronger than adenosine / 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.[2] 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.[3] 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 as temperature gradient gel electrophoresis.[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 (PCR). Although the temperature of DNA melting is not diagnostic in the technique, methods for estimating Tm are important for determining the appropriate temperatures to use in a protocol. DNA melting temperatures can also be used as a proxy for equalizing the hybridization strengths of a set of molecules, eg. the oligonucleotide probes of DNA microarrays. Annealing Annealing, in genetics, means for DNA or RNA to pair by hydrogen bonds to a complementary sequence, forming a double-stranded polynucleotide (See Nucleic acid hybridization). The term is often used to describe the binding of a DNA probe, or the binding of a primer to a DNA strand during a polymerase chain reaction (PCR). The term is also often used to describe the reformation (renaturation) of complementary strands that were separated by heat (thermally denatured). Proteins such as RAD52 can help DNA anneal. Methods for estimating melting temperatures Several formulas are used to calculate Tm values.[6][7] Some formulas are more accurate in predicting melting temperatures of DNA duplexes.[8] Nearest-neighbor method The nearest-neighbor method is one method used to predict melting temperatures of nucleic acid duplexes. Although GC content plays a large factor in the hybridization energy of double-stranded DNA, interactions between neighboring bases along the helix means that stacking energies are significant. The nearest-neighbor model accounts for this by considering adjacent bases along the backbone two-at-a-time.[1] Each of these has enthalpic and entropic parameters, the sums of which determine melting temperature according to the following equation: Tm =ΔH / ( ΔD +R ln(C1- C2/2)) R is the universal gas constant 1.987 cal / mol *K t). Standard enthalpies and entropies are negative for the annealing reaction and are assumed to be temperature independent. If C1 > > C2 then C2 can be neglected. Table 1. Unified nearest-neighbor parameters for DNA/DNA duplexes.[1] Note that the ΔH values are given in kilocalories per mole and that the ΔS values are given in calories per mole per kelvin.
See also * DNA
1. ^ a b c John SantaLucia Jr. (1998). "A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics". Proc. Natl. Acad. Sci. USA 95 (4): 1460–5. doi:10.1073/pnas.95.4.1460. [1]
* Tm calculations in OligoAnalyzer - Integrated DNA Technologies Retrieved from "http://en.wikipedia.org/"
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