DNA 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. For multiple copies of DNA molecules, the melting temperature (T_m) 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. The melting temperature depends on both the length of the molecule, and the specific nucleotide sequence composition of that molecule.

Applications of DNA denaturation

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. [cite journal |journal=Methods in Enzymology |title=Use of Ultravialet Absorbance-Temperature Profile for Determining the Guanine plus Cytosine Content of DNA |author=M. Mandel and J. Marmur |year=1968 |volume=12 |issue=2 | id=ISBN 978-0-12-181856-2 |pages=198–206] 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. [cite journal| title=The Phylogeny of the Hominoid Primates, as Indicated by DNA-DNA Hybridization| author=C.G. Sibley and J.E. Ahlquist| journal=Journal of Molecular Evolution| volume=20| pages=2–15| year=1984| doi=10.1007/BF02101980] 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. [cite journal| author=R.M. Myers, T. Maniatis, and L.S. Lerman| title=Detection and Localization of Single Base Changes by Denaturing Gradient Gel Electrophoresis| year=1987| journal=Methods in Enzymology| volume=155| pages=501–527| id=ISBN 978-0-12-182056-5] [cite journal| author=T. Po, G. Steger, V. Rosenbaum, J. Kaper, and D. Riesner| title=Double-stranded cucumovirus associated RNA 5: experimental analysis of necrogenic and non-necrogenic variants by temperature-gradient gel electrophoresis| journal=Nucleic Acids Research| year=1987| volume=15| issue=13| pages=5069–5083| doi=10.1093/nar/15.13.5069]

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 T_m 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.

Methods for estimating T_m

Several formulas are used to calculate T_m values. [ cite journal| author=Breslauer, K.J. et al.|year=1986| journal=Proc. Natl. Acad. Sci. USA.| title=Predicting DNA Duplex Stability from the Base Sequence|volume=83| pages=3746–3750| doi=10.1073/pnas.83.11.3746 [http://www.pubmedcentral.gov/picrender.fcgi?artid=323600&blobtype=pdf (pdf)] ] [ Rychlik, W. et al. (1990) "Nucleic Acids Res." 18, 6409-6412. ] Some formulas are more accurate in predicting melting temperatures of DNA duplexes. [cite journal| author=Owczarzy R., Vallone P.M., Gallo F.J., Paner T.M., Lane M.J. and Benight A.S | year=1997 |title=Predicting sequence-dependent melting stability of short duplex DNA oligomers | journal=Biopolymers |volume=44|pages=217–239 |doi=10.1002/(SICI)1097-0282(1997)44:3<217::AID-BIP3>3.0.CO;2-Y |doilabel=10.1002/(SICI)1097-0282(1997)44:3217::AID-BIP33.0.CO;2-Y [http://www3.interscience.wiley.com/cgi-bin/abstract/40314/ABSTRACT (pdf)] ]

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.cite journal| title=A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics| author= John SantaLucia Jr.| journal=Proc. Natl. Acad. Sci. USA| year=1998| volume=95| issue=4| pages=1460–5| doi=10.1073/pnas.95.4.1460 [http://www.pnas.org/cgi/content/full/95/4/1460] ] Each of these has enthalpic and entropic parameters, the sums of which determine melting temperature according to the following equation:

:$T\_mbox\{m\}=frac\{Delta\; H\; \}\{Delta\; S+R\; ln(C\_1-frac\{C\_2\}\{2\})\}-273.15\; ^circ\; mbox\{C\}$:where:$Delta\; H$ is the standard enthalpy and $Delta\; S$ is the standard entropy for formation of the duplex from two single strands,:$C\_1$ is the initial concentration of the single strand that is in excess (usually probe, primer),:$C\_2$ is the initial concentration of the complementary strand that is limiting (usually target),

:$R$ is the universal gas constant $left\; (mbox\{1.987\}frac\{mbox\{cal\{mbox\{mol\}\; cdot\; mbox\{K\; ight)$.

Standard enthalpies and entropies are negative for the annealing reaction and are assumed to be temperature independent. If $C\_1\; >>\; C\_2$ then $C\_2$ can be neglected.

Table 1. Unified nearest-neighbor parameters for DNA/DNA duplexes. ] Note that the $Delta\; H$ values are given in "kilo"calories per mole and that the $Delta\; S$ values are given in calories per mole per kelvin.

References

ee also

*DNA
*denaturation (biochemistry)
*hybridisation
*PCR
*Melting point
*Primer for calculations of Tm

External links

* [http://www.idtdna.com/analyzer/applications/Instructions/Default.aspx?AnalyzerDefinitions=true#MismatchMeltTemp T_m calculations in OligoAnalyzer] - Integrated DNA Technologies
* [http://www.biophp.org/minitools/melting_temperature/demo.php T_m calculation] - by bioPHP.org.
*http://www.promega.com/biomath/calc11.htm#disc
* [http://www.invitrogen.com/content/sfs/appendix/PCR_RTPCR/Primer%20Tm%20Calculations.pdf Invitrogen T_m calculation]
* [http://www.bioinformatics.org/annhyb AnnHyb Open Source software for T_m calculation using the Nearest-neighbour method]
* [http://www.sigmaaldrich.com/Brands/Sigma_Genosys/Custom_DNA/Key_Resources/Oligos_Melting_Temp.html Sigma-aldrich technical notes]
* [http://fokker.wi.mit.edu/primer3/input-help.htm#PRIMER_TM Primer3 calculation]