Analysis of thermal melting curves

T(m) is defined as Temperature of melting or, more accurately, as temperature of midtransition. This term is often used for nucleic acids (DNA and RNA, oligonucleotides and polynucleotides). A thermal denaturation experiment determines the stability of the secondary structure of a DNA or RNA and aids in the choice of the sequences for antisense oligomers or PCR primers. Beyond a simple numerical value (the T(m)), a thermal denaturation experiment, in which the folded fraction of a structure is plotted vs. temperature, yields important thermodynamic information. We present the classic problems encountered during these experiments and try to demonstrate that a number of useful pieces of information can be extracted from these experimental curves.     

Theory of DNA melting curves

Exact algorithms for the calculation of melting curves of heterogeneous DNA with N base pairs apparently require computer time proportional to N². However, it is shown that a decomposition of the loop entropy factor into a sum of I exponential functions (1) gives an extremely accurate approximation to the loop entropy factor for small values of I and (2) makes the computer time for the exact algorithms proportional to I·N. In effect, exact results for melting curves and lengths of helix or coil stretches are obtained with computer time comparable to that required for the Frank-Kamenetskii approximation. The remarkable accuracy of the latter for the fraction of helical content (errors of 0.01–0.05) is confirmed, but appreciably larger errors are found for the lengths of helix or coil stretches (typical errors of 30–100%).     

Helix-coil transitions in nucleoprotein: Computer-generated curves for independent melting of double-stranded regions with fixed ends

Helix-coil transitions in nucleoprotein at low salt concentrations are known to be characterized by two phases of the process: independent melting of uncomplexed “naked” regions without rearrangement of proteins, followed, at higher temperatures, by melting of complexed DNA. Blocking at the ends of these regions increases their thermal stability and three is a shift of 10–20°C in t_m of the melting profiles. In this study the basic assumption is that the loop entropy effect is mainly responsible for such stabilization. Calculations are made using conventional h-c transition theory for a system of independently melted segments with fixed ends. Segments are either homosize or have randomly distributed lengths. Calculated melting curves are used to obtain t_m, and transition width-dependence on segment length (or average length when randomly distributed) and on the nucleation parameter σ. Base-pair heterogeneity is taken into account by averaging over different base-pair distributions in the individual segments, using Gaussian distribution around the overall (G+C)-content. It is shown that this causes only an additional widening of the transition but no additional t_m shift. Comparison is made with similar systems in the literature. The main conclusion drawn is that the treatment proposed may be useful for analysis of the lower temperature melting phase in nucleoprotein at low counterion concentrations. It may be used as an independent method to reveal the features of nucleoprotein structure.     

Determination of melting temperature and temperature melting range for DNA with multi-peak differential melting curves

Many factors that change the temperature position and interval of the DNA helix–coil transition often also alter the shape of multi-peak differential melting curves (DMCs). For DNAs with a multi-peak DMC, there is no agreement on the most useful definition for the melting temperature, T_m, and temperature melting width, ΔT, of the entire DNA transition. Changes in T_m and ΔT can reflect unstable variation of the shape of the DMC as well as alterations in DNA thermal stability and heterogeneity. Here, experiments and computer modeling for DNA multi-peak DMCs varying under different factors allowed testing of several methods of defining T_m and ΔT. Indeed, some of the methods give unreasonable “jagged” T_m and ΔT dependences on varying relative concentration of DNA chemical modifications (r_b), [Na⁺], and GC content. At the same time, T_m determined as the helix–coil transition average temperature, and ΔT, which is proportional to the average absolute temperature deviation from this temperature, are suitable to characterize multi-peak DMCs. They give smoothly varying theoretical and experimental dependences of T_m and ΔT on r_b, [Na⁺], and GC content. For multi-peak DMCs, T_m value determined in this way is the closest to the thermodynamic melting temperature (the helix–coil transition enthalpy/entropy ratio).     

Fine structure of DNA melting curves.

Theoretical calculations predict that the differential melting curves for random polynucleotide sequences having lengths up to several tens of thousands of base pairs have a clear-cut fine structure. This structure appears in the form of multiple narrow peaks 0.3–0.4°C wide on the bell shaped main curve. The differential melting curves have different shapes for different specific sequences. The theory also predicts the disappearance of the fine structure when the length of the sequence increases and when circular, covalently closed DNA is considered instead of the open structure. The predictions of the theory were confirmed by the measurements of differential melting curves for open and covalently closed circular forms of DNA for PM2 phage (N = 10⁴ base pairs) and also for other phage DNA's of different length: T7 (N = 3.8 × 10⁴); S_D (N = 9.2 × 10⁴); T2 (N = 17 × 10⁴). It was shown that the effect of fine structure results mainly from the cooperative melting out of DNA regions 300–500 base pairs long.     

Phase transitions in nuclei and chromatin

Changes in the volume of rat liver nuclei have been monitored as a function of modifications in ionic environment (from 0 to 20 mM), temperature (from 4 to 37°C) and pH (from 1 to 8). An abrupt reduction of nuclear volume occurred with increasing ion concentration, this contraction being more pronounced with bivalent (either Ca²⁺ or Mg²⁺) than with monovalent (either Na⁺ or K⁺) cations. The lowering of pH produced a similar effect. Parallel changes in chromatin structure took place at the same time as phase-like transitions. Atomic absorption spectroscopy allowed determination of free and nuclei-bound ions, pointing to the presence of a sizeable number of free binding sites for chromatin-DNA even within intact nuclei. DNA-phosphate sites appear to be neutralized by ions strictly according to the size of the electric charge and polyelectrolyte theory. Partial digestion (by micrococcal nuclease) or simple breaks (by chemical carcinogens) of the chromatin-DNA fiber caused respectively elimination or reduction of the abrupt volume changes in the intact nuclei. The apparent role of chromatin structure versus nuclear matrix in determining the shape and volume of intact nuclei is briefly discussed.     

Piecewise exponential survival curves with smooth transitions

Several models of a population survival curve composed of two piecewise exponential distributions are developed. In one formulation the hazard rate changes at a point that is an unobservable random variable that varies between individuals. The population hazard function may decrease with age even when all individuals' hazards are increasing. In a second formulation, the population hazard function is modeled directly. Several models are fit to the survival history of a cohort of 5751 highly inbred male Drosophila melanogaster and the British coal mining disaster data.     

Phase transitions of polymerizable phospholipids

Polymerizable lipids have received considerable attention in the last ten years as polymerization of lipids in vesicle systems is a possibility to increase the stability of lipid bilayers. Lipids with various polymerizable groups have been synthesized in the last years. This paper is focussed on those lipids which are closely related to natural phospholipids, i.e. molecules which have two hydrophobic chains and a head group containing a phosphate moiety. The phase behaviour of polymerizable phospholipids as lipid monomers and in the polymerized state is reviewed and discussed.     

Singular value decomposition of 3-D DNA melting curves reveals complexity in the melting process

The thermal denaturation of synthetic deoxypolynucleotides of defined sequence was studied by a three dimensional melting technique in which complete UV absorbance spectra were recorded as a function of temperature. The results of such an experiment defined a surface bounded by absorbance, wavelength, and temperature. A matrix of the experimental data was built, and analyzed by the method of singular value decomposition (SVD). SVD provides a rigorous, model-free analytical tool for evaluating the number of significant spectral species required to account for the changes in UV absorbance accompany-ing the duplex – to – single strand transition. For all of the polynucleotides studied (Poly dA – Poly dT; [Poly (dAdT)]₂; Poly dG – Poly dC; [Poly(dGdC)]₂), SVD indicated the existence of at least 4 – 5 significant spectral species. The DNA melting transition for even these simple repeating sequences cannot, therefore, be a simple two-state process. The basis spectra obtained by SVD analysis were found to be unique for each polynucleotide studied. Differential scanning calorimetry was used to obtain model free estimates for the enthalpy of melting for the polynucleotides studied, with results in good agreement with previously published values. Received: 16 April 1997 / Accepted: 9 July 1997     

Single nucleotide polymorphism genotyping using allele-specific PCR and fluorescence melting curves

We present a PCR method for identification of single nucleotide polymorphisms (SNPs), using allele-specific primers designed for selective amplification of each allele. Matching the SNP at the 3' end of the forward or reverse primer, and additionally incorporating a 3' mismatch to prevent amplification of the incorrect allele, results in selectivity of the allele-specific primers. DNA melting analysis with fluorescent SYBR Green affords detection of the PCR products. By incorporating a GC-rich sequence into one of the two allele-specific primers to increase the melting temperature, both alleles can be measured simultaneously at their respective melting temperatures. Applying the DNA melting analysis to SNPs in ApoE and ABCA1 yielded results identical to those obtained with other genotyping methods. This provides a cost-effective, high-throughput method for amplification and scoring of SNPs.     

Phase response curves for social entrainment

Summary Phase shifts in free-running activity rhythms of male golden hamsters,Mesocricetus auratus, often occur when they establish a new territory and home after a cage change. Similar shifts also often occur after pairs of animals interact with each other for half an hour. When these events take place during the middle of the hamsters' subjective day, they produce phase advances: when late in the subjective night, they produce phase delays. Repeated social interactions at the same time of day can entrain activity rhythms in a way consistent with the shape of the phase response curves. Not all individuals become entrained, as is predictable from the modest amplitude of the phase response curve. The effects of social interactions and of other disturbances may be mediated through an oscillator phased by general arousal. The present findings have implications for the interpretation of drug-induced changes in biological rhythms.     

Thermophoretic melting curves quantify the conformation and stability of RNA and DNA

Measuring parameters such as stability and conformation of biomolecules, especially of nucleic acids, is important in the field of biology, medical diagnostics and biotechnology. We present a thermophoretic method to analyse the conformation and thermal stability of nucleic acids. It relies on the directed movement of molecules in a temperature gradient that depends on surface characteristics of the molecule, such as size, charge and hydrophobicity. By measuring thermophoresis of nucleic acids over temperature, we find clear melting transitions and resolve intermediate conformational states. These intermediate states are indicated by an additional peak in the thermophoretic signal preceding most melting transitions. We analysed single nucleotide polymorphisms, DNA modifications, conformational states of DNA hairpins and microRNA duplexes. The method is validated successfully against calculated melting temperatures and UV absorbance measurements. Interestingly, the methylation of DNA is detected by the thermophoretic amplitude even if it does not affect the melting temperature. In the described setup, thermophoresis is measured all-optical in a simple setup using a reproducible capillary format with only 250 nl probe consumption. The thermophoretic analysis of nucleic acids shows the technique's versatility for the investigation of nucleic acids relevant in cellular processes like RNA interference or gene silencing.     

高分辨率熔解曲线法的非标记探针基因分型技术

PCR反应中利用荧光检测技术对已知位点进行基因分型时常采用荧光标记的寡核苷酸做探针。近年来新兴起的高分辨率熔解曲线技术可以采用非标记的探针对已知位点的SNP(single nucleotide polymorphism)或突变进行基因分型研究。采用非标记探针法对已知位点的基因分型研究具有廉价、快速、简便等特点,因此被大量应用在和疾病、形状等相关的一些多肽位点的研究中。本文较详细地介绍该技术的基本原理和实验中的注意事项。     

High throughput measurement of duplex, triplex and quadruplex melting curves using molecular beacons and a LightCycler

We have used oligonucleotides containing molecular beacons to determine melting profiles for intramolecular DNA duplexes, triplexes and quadruplexes (tetraplexes). The synthetic oligonucleotides used in these studies contain a fluorophore (fluorescein) and quencher (methyl red) attached either to deoxyribose or to the 5 position of dU. In the folded DNA structures the fluorophore and quencher are in close proximity and the fluorescence is quenched. When the structures melt, the fluorophore and quencher are separated and there is a large increase in fluorescence. These experiments were performed in a Roche LightCycler; this requires small amounts of material (typically 4 pmol oligonucleotide) and can perform 32 melting profiles in parallel. We have used this technique to compare the stability of triplexes containing different base analogues and to confirm the selectivity of a triplex-binding ligand for triplex, rather than duplex, DNA. We have also compared the melting of inter- and intramolecular quadruplexes.