Predicted Melting Temperature Research Articles

A set of nearest neighbor parameters for predicting the enthalpy change of RNA secondary structure formation

A complete set of nearest neighbor parameters to predict the enthalpy change of RNA secondary structure formation was derived. These parameters can be used with available free energy nearest neighbor parameters to extend the secondary structure prediction of RNA sequences to temperatures other than 37°C. The parameters were tested by predicting the secondary structures of sequences with known secondary structure that are from organisms with known optimal growth temperatures. Compared with the previous set of enthalpy nearest neighbor parameters, the sensitivity of base pair prediction improved from 65.2 to 68.9% at optimal growth temperatures ranging from 10 to 60°C. Base pair probabilities were predicted with a partition function and the positive predictive value of structure prediction is 90.4% when considering the base pairs in the lowest free energy structure with pairing probability of 0.99 or above. Moreover, a strong correlation is found between the predicted melting temperatures of RNA sequences and the optimal growth temperatures of the host organism. This indicates that organisms that live at higher temperatures have evolved RNA sequences with higher melting temperatures.

Genotyping of Ovine Prion Protein Gene (PRNP) Variants by PCR with Melting Curve Analysis,

Scrapie is the transmissible spongiform encephalopathy in sheep. Because genetic variants of the ovine PrP gene (PRNP) can be associated with disease risk, the European Union initiated programs to eradicate high-risk PRNP genotypes from sheep livestock. For this purpose, reliable and cost-effective genotyping is needed. We amplified DNA to cover the 3 risk codons in exon 3 encoding amino acids 136, 154, and 171. Amplicons were mixed with dye-labeled probe sets, and melting curves were recorded in a LightCycler by use of color and temperature multiplexing. Probe design was based on thermodynamic calculations to ensure unequivocal results for the 3 codons of interest, taking the additional F141 and T137 sequence variants into account. The fluorescence resonance energy transfer (FRET) method, when compared with sequencing, gave exactly the predicted melting temperatures for all possible genotypes. When we validated the method with samples from official certification programs, it showed completely matching results. Turnaround time was approximately 5 h after receipt of a whole-blood sample. The method detected the rare sequence variants T137 and F141, which were clearly distinguishable from the other known genotypes by melting curve analysis. One scrapie sheep was ARR/ARR, which is considered the haplotype with the lowest risk. The FRET-based PRNP genotyping method for sheep is rapid and can differentiate all genotypes at each locus in 1 capillary. The assay is fast and has lower costs than restriction fragment length polymorphism analysis or sequencing.

Affinity capillary electrophoresis of DNA for detection of single-nucleotide polymorphisms and point mutations: Comprehensive study for optimization of the weak affinity

A single-stranded DNA and its point mutant can be separated with affinity capillary electrophoresis (ACE) in which an appropriate ligand DNA is used as a pseudo-stationary affinity phase. In this paper, we systematically examine the effects of ligand sequence, capillary temperature, and cation concentration on the ACE separation quality, which is quantitatively evaluated in terms of resolution and peak height. For fine tuning of the affinity, control of MgCl 2 concentration and insertion of a spacer sequence into the ligand DNA are more effective than control of the capillary temperature. For design of the ligand DNA, a simple strategy is proposed, based on the prediction of melting temperature. This strategy was tested with eleven different sample sequences. All of them were separated under the same conditions (250 μM MgCl 2 and 25 °C), and nine cases of them had satisfactory separation qualities.

Oligonucleotide properties determination and primer designing: a critical examination of predictions

Precise prediction of melting temperature (T(m)), secondary structures and design of oligonucleotides determine the efficiency and success of experimentation in molecular biology. Availability of a plethora of software and the users unawareness about their limitations compromises the accuracy and reliability of the predictions. Comparative analysis of 56 modules was done for T(m) prediction using a large set of oligonucleotide sequences spanning the whole range of GC-content and length. Allawi module of the calculator 'MELTING', Nearest Neighbor (NN) of oligo calculator (McLab), NN of T(m) Calculation for Oligos (Biomath Calculator, Promega) and HYTHER provided the most precise T(m) predictions. A model has also been proposed to calculate the optimum annealing temperature integrating the already reported formulations. Secondary structure predictions of oligonucleotides reveal a large number of structures in contrast to the experimental observations. Of the 11 primer designing tools evaluated, Primer 3 and WebPrimer performed the best for the AT-rich templates, Exon Primer for AT = GC templates, and Primer Design Assistant, Primer3 and Primer Quest for GC-rich templates. This study provides optimal choice for application to the user, increasing the success of a variety of experimentations, especially those that have high-throughput and complex assay designs. db@igib.res.in The details of the oligonucleotides and of the different modules of T(m) prediction considered for the study are provided as Supplementary Information, available at Bioinformatics online.

DnaMATE: a consensus melting temperature prediction server for short DNA sequences

An accurate and robust large-scale melting temperature prediction server for short DNA sequences is dispatched. The server calculates a consensus melting temperature value using the nearest-neighbor model based on three independent thermodynamic data tables. The consensus method gives an accurate prediction of melting temperature, as it has been recently demonstrated in a benchmark performed using all available experimental data for DNA sequences within the length range of 16–30 nt. This constitutes the first web server that has been implemented to perform a large-scale calculation of melting temperatures in real time (up to 5000 DNA sequences can be submitted in a single run). The expected accuracy of calculations carried out by this server in the range of 50–600 mM monovalent salt concentration is that 89% of the melting temperature predictions will have an error or deviation of <5°C from experimental data. The server can be freely accessed at . The standalone executable versions of this software for LINUX, Macintosh and Windows platforms are also freely available at the same web site. Detailed further information supporting this server is available at the same web site referenced above.

A potential model for the study of ices and amorphous water: TIP4P/Ice

The ability of several water models to predict the properties of ices is discussed. The emphasis is put on the results for the densities and the coexistence curves between the different ice forms. It is concluded that none of the most commonly used rigid models is satisfactory. A new model specifically designed to cope with solid-phase properties is proposed. The parameters have been obtained by fitting the equation of state and selected points of the melting lines and of the coexistence lines involving different ice forms. The phase diagram is then calculated for the new potential. The predicted melting temperature of hexagonal ice (Ih) at 1 bar is 272.2 K. This excellent value does not imply a deterioration of the rest of the properties. In fact, the predictions for both the densities and the coexistence curves are better than for TIP4P, which previously yielded the best estimations of the ice properties.

Rapid Species Identification Within theMycobacterium chelonae–abscessusGroup by High-Resolution Melting Analysis ofhsp65PCR Products

Polymerase chain reaction (PCR) amplification of the heat shock protein 65 (hsp65) gene followed by high-resolution melting analysis with LCGreen I (Idaho Technology, Salt Lake City, UT) was used to differentiate the mycobacteria species Mycobacterium chelonae, Mycobacterium abscessus, and Mycobacterium immunogenum in less than 20 minutes. A 105-base-pair amplicon that clustered the different species by predicted melting temperature was found from available GenBank hsp65 sequences. We identified 24 clinical isolates within the M chelonae-abscessus group by proximal 16S ribosomal RNA and hsp65 gene sequencing. Rapid-cycle PCR followed by high-resolution melting analysis clustered these samples into the following groups: M abscessus, 12; M abscessus sequence variant, 2; M chelonae, 7; unexpected M chelonae sequence variant, 1; and M immunogenum, 2. The M chelonae variant had a single base change not found in reported GenBank sequences. Advantages of the method include speed, low risk of amplicon contamination (closed-tube), and no need for separation steps (sequencing, electrophoresis, high-performance liquid chromatography) or real-time monitoring.

Stekel, D. Microarray bioinformatics

Stekel, D. Microarray bioinformatics

Melting of lithium hydride under pressure.

We have computed the melting line of lithium hydride up to 200 GPa using the two-phase simulation technique coupled with first-principles molecular dynamics. Our predicted melting temperature at high pressures varies slowly with compression, ranging from 2000 to 2450 K at 50-200 GPa pressures. The compressed fluid close to the melting line retains the ionic character of the low pressure molten state, while at higher temperatures dynamical hydrogen clustering processes are observed, which are accompanied by changes in the electronic structure.

OligoDesign: Optimal design of LNA (locked nucleic acid) oligonucleotide capture probes for gene expression profiling.

We report the development of new software, OligoDesign, which provides optimal design of LNA (locked nucleic acid) substituted oligonucleotides for functional genomics applications. LNAs constitute a novel class of bicyclic RNA analogs having an exceptionally high affinity and specificity toward their complementary DNA and RNA target molecules. The OligoDesign software features recognition and filtering of the target sequence by genome-wide BLAST analysis in order to minimize cross-hybridization with non-target sequences. Furthermore it includes routines for prediction of melting temperature, self-annealing and secondary structure for LNA substituted oligonucleotides, as well as secondary structure prediction of the target nucleotide sequence. Individual scores for all these properties are calculated for each possible LNA oligonucleotide in the query gene and the OligoDesign program ranks the LNA capture probes according to a combined fuzzy logic score and finally returns the top scoring probes to the user in the output. We have successfully used the OligoDesign tool to design a Caenorhabditis elegans LNA oligonucleotide microarray, which allows monitoring of the expression of a set of 120 potential marker genes for a variety of stress and toxicological processes and toxicologically relevant pathways. The OligoDesign program is freely accessible at http://lnatools.com/.

Defect melting models for cubic lattices and universal laws for melting temperatures

We set up simple harmonic lattice models for elastic fluctuations in bcc and fcc lattices and the excitation of dislocations and disclinations. From these we derive, in the lowest approximation, universal formulas which predict melting temperatures in good agreement with the experiments. This new theory is more precise than Lindemann's rule by factor 2, and more predictive, since the size of the Lindemann number has to be fixed by experiments. In addition, our theory allows for systematic improvements.

ProbeBase: an online resource for rRNA-targeted oligonucleotide probes.

Ribosomal RNA-(rRNA)-targeted oligonucleotide probes are widely used for culture-independent identification of microorganisms in environmental and clinical samples. ProbeBase is a comprehensive database containing more than 700 published rRNA-targeted oligonucleotide probe sequences (status August 2002) with supporting bibliographic and biological annotation that can be accessed through the internet at http://www.probebase.net. Each oligonucleotide probe entry contains information on target organisms, target molecule (small- or large-subunit rRNA) and position, G+C content, predicted melting temperature, molecular weight, necessity of competitor probes, and the reference that originally described the oligonucleotide probe, including a link to the respective abstract at PubMed. In addition, probes successfully used for fluorescence in situ hybridization (FISH) are highlighted and the recommended hybridization conditions are listed. ProbeBase also offers difference alignments for 16S rRNA-targeted probes by using the probe match tool of the ARB software and the latest small-subunit rRNA ARB database (release June 2002). The option to directly submit probe sequences to the probe match tool of the Ribosomal Database Project II (RDP-II) further allows one to extract supplementary information on probe specificities. The two main features of probeBase, 'search probeBase' and 'find probe set', help researchers to find suitable, published oligonucleotide probes for microorganisms of interest or for rRNA gene sequences submitted by the user. Furthermore, the 'search target site' option provides guidance for the development of new FISH probes.

A generalized equation for the prediction of melting temperatures of homopolymers and copolymers

AbstractA generalized equation was derived to calculate the melting temperatures of homopolymers and copolymers. The Gibbs‐Thomson equation for homopolymers and a modified application to copolymers were derived using the proposed equation. The melting temperature Tm0 in the Flory equation corresponds to the melting temperature TmC,∞ of copolymer crystals with stems of infinite length. Also, TmC,n*, the melting temperature for copolymer crystals with stems containing the maximum possible number of structural units, n*, should be used instead of Tm0 as the basis of supercooling in crystallization. The proposed equation shows good agreement with experimental data for α‐alkene‐ethylene homogeneous copolymers.

Predicting melting temperature (Tm) of oligoribonucleotide duplex by neural network

AbstractA straightforward method to predict melting temperatures of RNA duplexes by artificial neural network is developed. The network consists of three layers in which there are 12 input layer units (10 nearest neighbors and two terminals), two hidden layer units and one output layer unit (melting temperature). It is found that the predicted data for the test set agree with the measured data quite well (AD = 3.7 K, R2 = 0.9359, SEP = 4.17 for NN1; AD = 2.85 K, R2 = 0.8843, SEP = 3.43 for NN2). This method omits the determination of thermodynamic parameters and therefore is a simpler way to predict the stability of RNA duplex. Copyright © 2002 John Wiley &amp; Sons, Ltd.

Structure-property correlation and prediction of melting temperature of RNA duplexes.

Structure-property correlation and prediction of melting temperature of RNA duplexes.

Theoretic information approach to protein stabilization by solvent engineering

AbstractA novel approach based on molecular thermodynamics and the information theory is proposed to quantify the influence of water‐miscible additives on protein stability. According to the two‐state mechanism of inactivation, solvent effects are described in terms of perturbation of the equilibrium between the folded and unfolded protein forms. The model provides the dependence of the protein's melting temperature on the additive concentration. Effects of the latter are accounted for by an empirical parameter related to the free energy of transfer of the protein from the pure to the mixed solvent. The model was tested using experimental data relative to the influence of hydroxylic and aminoacidic additives on the thermal unfolding of hen egg lysozyme and erythrocyte carbonic anhydrase. Fitting parameters were correlated in terms of a theoretic information index characterizing the additive's molecule and incorporating an atomic‐composition term and a topological contribution. Model calculations agreed very well with experimental data, suggesting that the molecular information content of the additive can be used effectively to correlate solvent‐induced perturbations of stability. The procedure was also used to predict melting temperatures in systems containing binary mixtures of additives and to reconstruct thermal unfolding curves in the different media.

Relative melting temperatures of RNase HI mutant proteins from MD simulation/free energy calculations

The melting temperatures of three RNase HI mutants were determined from molecular dynamics (MD) simulation/free energy calculations. MD simulations and free energy calculations were carried out for the protein in water with all degrees of freedom and long-range Coulomb interactions included. The predicted melting temperatures were in good agreement with experimental values without the need of any adjustable parameters.

Molecular dynamics study of the plastic-crystalline phase transition of tetraphosphorus triselenide

A seven-site atom-atom potential model has been developed to describe the interactions between tetraphosphorus triselenide (P4Se3) molecules. When used in molecular dynamics calculations the model predicts a transition to an orientationally disordered phase at a temperature close to that of the experimentally observed disordering transition. The predicted melting temperature of the model also is in good agreement with experiment. Below the transition, reorientation of the P4Se3 molecules is of purely librational character. Above the transition, the molecules rotate about all three principal axes, but end-over-end rotation of the molecule is favoured relative to spinning motion around the threefold axis; the rotation becomes more isotropic as the temperature increases. Orientational disordering is shown to be accompanied by a transition to a hexagonal-close-packed structure. The positions and intensities of lines in the X-ray scattering pattern of the model system suggest that the structure of the disorder...

Melting of Aromatic Compounds: Molecular Dynamics Simulations

AbstractWe have used constant pressure molecular dynamics calculations to explore the behavior at various temperatures of two molecular crystals: benzene and a brominated phenyl compound. We observed a melting transition by heating the crystals from a low temperature. In the case of benzene, we performed one heating run of about 1 ns and obtained agreement with the experimental melting point to within some 8%. We have also simulated the melting of a more complex molecular crystal that contains bromine and phenyl groups. We performed four heating runs, with different rates of heating. For total simulation times of about 100, 220, 770, and 1 I50ps, the heating runs predicted melting temperatures that differed from the experimental melting temperature by 53%, 33%, 25%, and 9% respectively.

Solid-solid transition and solubility of even n-alkanes

Abstract Ksiazczak, A., Moorthi, K. and Nagata, I., 1994. Solid-solid transition and solubility of even n-alkanes. Fluid Phase Equilibria, 95: 15-29. Solubility data for long-chain n-alkanes in shorter-chain alkanes have been used to predict the melting temperatures of the polymorphic phase stable at lower temperatures. In the case of alkanes having no polymorphic phases (hexadecane and octadecane) the predicted melting temperatures are consistent with the conventional melting temperature of the pure alkanes. The predicted melting temperatures of alkanes possessing polymorphic phases are consistent with the transition temperatures of pure alkanes (tetracosane, hexacosane, octacosane, dotriacontane and hexatriacontane). The transition between the polymorphic phases of pure even n-alkanes proceeds by the transitional liquid state at the melting temperatures of the solid phases stable at lower temperatures.