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).     

A method for the ligation of DNA following isolation from low melting temperature agarose

A rapid, simple, and reliable method is presented for the isolation and subsequent ligation of DNA from agarose gels. The technique involves the use of low melting temperature agarose, but with the inclusion of bovine serum albumin or gelatin to the ligation reaction.     

Direct measurement of DNA by means of AFM.

We have demonstrated that the electrostatic stretch-and-positioning method is useful for the analysis of a long DNA molecule by means of atomic force microscopy (AFM). DNA molecules were stretched parallel to the field line, and immobilized onto the aluminum electrodes patterned on a glass plate. Through AFM observation, we confirmed the immobilization of individual DNA molecules, not aggregate.     

Direct DNA sequencing of PCR amplified genomic DNA by the Maxam-Gilbert method

We present a method to directly sequence PCR amplification products utilizing the chemical method of Maxam and Gilbert. This procedure yields clearly readable DNA sequences of 100-400 base pairs in length derived from human genomic DNA in four days' time.     

Influence of formaldehyde on the melting temperature of DNA]

It has been shown that formaldehyde has no marked physical effect upon DNA resulting in lowering of its melting temperature. The effect of lowering of DNA melting temperature observed earlier by other authors resulted from the process of unwinding of DNA due to chemical reactions of formaldehyde with reactive base groups.     

Direct measurement of the mechanical properties of lipid phases in supported bilayers

Biological membranes define not only the cell boundaries but any compartment within the cell. To some extent, the functionality of membranes is related to the elastic properties of the lipid bilayer and the mechanical and hydrophobic matching with functional membrane proteins. Supported lipid bilayers (SLBs) are valid biomimetic systems for the study of membrane biophysical properties. Here, we acquired high-resolution topographic and quantitative mechanics data of phase-separated SLBs using a recent atomic force microscopy (AFM) imaging mode based on force measurements. This technique allows us to quantitatively map at high resolution the mechanical differences of lipid phases at different loading forces. We have applied this approach to evaluate the contribution of the underlying hard support in the determination of the elastic properties of SLBs and to determine the adequate indentation range for obtaining reliable elastic moduli values. At ~200 pN, elastic forces dominated the force-indentation response and the sample deformation was <20% of the bilayer thickness, at which the contribution of the support was found to be negligible. The obtained Young's modulus (E) of 19.3 MPa and 28.1 MPa allowed us to estimate the area stretch modulus (k(A)) as 106 pN/nm and 199 pN/nm and the bending stiffness (k(c)) as 18 k(B)T and 57 k(B)T for the liquid and gel phases, respectively.     

Direct electrochemical sensor for fast reagent-free DNA detection

A novel reagentless direct electrochemical DNA sensor has been developed using ultrathin films of the conducting polymer polypyrrole doped with an oligonucleotide probe. Our goal was to develop a prototype electrochemical DNA sensor for detection of a biowarfare pathogen, variola major virus. The sensor has been optimized for higher specificity and sensitivity. It was possible to detect 1.6 fmol of complementary oligonucleotide target in 0.1 ml in seconds by using chronoamperometry. The sensitivity of the developed sensor is comparable to indirect electrochemical DNA sensors, which use electrochemical labels and reagent-intensive amplification. The developed sensing electrode is reusable, highly stable and suitable for storage in solution or in dry state.     

DNA melting profiles from a matrix method

In this article we give a new method for the calculation of DNA melting profiles. Based on the matrix formulation of the DNA partition function, the method relies for its efficiency on the fact that the required matrices are very sparse, essentially reducing matrix multiplication to vector multiplication and thus making the computer time required to treat a DNA molecule containing N base pairs proportional to N(2). A key ingredient in the method is the result that multiplication by the inverse matrix can also be reduced to vector multiplication. The task of calculating the melting profile for the entire genome is further reduced by treating regions of the molecule between helix-plateaus, thus breaking the molecule up into independent parts that can each be treated individually. The method is easily modified to incorporate changes in the assignment of statistical weights to the different structural features of DNA. We illustrate the method using the genome of Haemophilus influenzae.     

Target concentration dependence of DNA melting temperature on oligonucleotide microarrays

The design of microarrays is currently based on studies focusing on DNA hybridization reaction in bulk solution. However, the presence of a surface to which the probe strand is attached can make the solution‐based approximations invalid, resulting in sub‐optimum hybridization conditions. To determine the effect of surfaces on DNA duplex formation, the authors studied the dependence of DNA melting temperature (T_m) on target concentration. An automated system was developed to capture the melting profiles of a 25‐mer perfect‐match probe–target pair initially hybridized at 23°C. Target concentrations ranged from 0.0165 to 15 nM with different probe amounts (0.03–0.82 pmol on a surface area of 10¹⁸ Ų), a constant probe density (5 × 10¹² molecules/cm²) and spacer length (15 dT). The authors found that T_m for duplexes anchored to a surface is lower than in‐solution, and this difference increases with increasing target concentration. In a representative set, a target concentration increase from 0.5 to 15 nM with 0.82 pmol of probe on the surface resulted in a T_m decrease of 6°C when compared with a 4°C increase in solution. At very low target concentrations, a multi‐melting process was observed in low temperature domains of the curves. This was attributed to the presence of truncated or mismatch probes. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Preliminary dielectric measurement and analysis protocol for determining the melting temperature and binding energy of short sequences of DNA in solution

Measurement of the real dielectric constant of bulk buffer solutions containing short sequences of DNA as a function of temperature through the DNA melting or denaturiztion transition can be used to determine melting temperatures, T(m), and to estimate the binding energy of the complimentary strands. We describe a preliminary dielectric measurement and analysis protocol to determine these parameters and its application to two known short sequences. The relative real dielectric constant for the bulk solutions was determined over the frequency range of 50 Hz-20 kHz and temperature range of <40-65 degrees C. The measurements were performed on dilute solutions and utilized low electric field strengths. Based on fits to the data by modified sigmoid functions, the melting temperatures, width of transition, and binding energy for the two sequences in solution were estimated. It was observed that the order of the transition appeared to be second order. The results were then compared against predictions of a number of models from the literature that provide theoretical estimates for the melting temperatures of known short sequences of DNA.     

A fractional programming approach to efficient DNA melting temperature calculation

MOTIVATION: In a wide range of experimental techniques in biology, there is a need for an efficient method to calculate the melting temperature of pairings of two single DNA strands. Avoiding cross-hybridization when choosing primers for the polymerase chain reaction or selecting probes for large-scale DNA assays are examples where the exact determination of melting temperatures is important. Beyond being exact, the method has to be efficient, as these techniques often require the simultaneous calculation of melting temperatures of up to millions of possible pairings. The problem is to simultaneously determine the most stable alignment of two sequences, including potential loops and bulges, and calculate the corresponding melting temperature. RESULTS: As the melting temperature can be expressed as a fraction in terms of enthalpy and entropy differences of the corresponding annealing reaction, we propose to use a fractional programming algorithm, the Dinkelbach algorithm, to solve the problem. To calculate the required differences of enthalpy and entropy, the Nearest Neighbor model is applied. Using this model, the substeps of the Dinkelbach algorithm in our problem setting turn out to be calculations of alignments which optimize an additive score function. Thus, the usual dynamic programming techniques can be applied. The result is an efficient algorithm to determine melting temperatures of two DNA strands, suitable for large-scale applications such as primer or probe design. AVAILABILITY: The software is available for academic purposes from the authors. A web interface is provided at http://www.zaik.uni-koeln.de/bioinformatik/fptm.html     

Comparison of different melting temperature calculation methods for short DNA sequences

MOTIVATION: The overall performance of several molecular biology techniques involving DNA/DNA hybridization depends on the accurate prediction of the experimental value of a critical parameter: the melting temperature Tm. Till date, many computer software programs based on different methods and/or parameterizations are available for the theoretical estimation of the experimental Tm value of any given short oligonucleotide sequence. However, in most cases, large and significant differences in the estimations of Tm were obtained while using different methods. Thus, it is difficult to decide which Tm value is the accurate one. In addition, it seems that most people who use these methods are unaware about the limitations, which are well described in the literature but not stated properly or restricted the inputs of most of the web servers and standalone software programs that implement them. RESULTS: A quantitative comparison on the similarities and differences among some of the published DNA/DNA Tm calculation methods is reported. The comparison was carried out for a large set of short oligonucleotide sequences ranging from 16 to 30 nt long, which span the whole range of CG-content. The results showed that significant differences were observed in all the methods, which in some cases depend on the oligonucleotide length and CG-content in a non-trivial manner. Based on these results, the regions of consensus and disagreement for the methods in the oligonucleotide feature space were reported. Owing to the lack of sufficient experimental data, a fair and complete assessment of accuracy for the different methods is not yet possible. Inspite of this limitation, a consensus Tm with minimal error probability was calculated by averaging the values obtained from two or more methods that exhibit similar behavior to each particular combination of oligonucleotide length and CG-content class. Using a total of 348 DNA sequences in the size range between 16mer and 30mer, for which the experimental Tm data are available, we demonstrated that the consensus Tm is a robust and accurate measure. It is expected that the results of this work would be constituted as a useful set of guidelines to be followed for the successful experimental implementation of various molecular biology techniques, such as quantitative PCR, multiplex PCR and the design of optimal DNA microarrays.     

Direct measurement of osmotic pressure of glycosaminoglycan solutions by membrane osmometry at room temperature

Articular cartilage is a hydrated soft tissue composed of negatively charged proteoglycans fixed within a collagen matrix. This charge gradient causes the tissue to imbibe water and swell, creating a net osmotic pressure that enhances the tissue's ability to bear load. In this study we designed and utilized an apparatus for directly measuring the osmotic pressure of chondroitin sulfate, the primary glycosaminoglycan found in articular cartilage, in solution with varying bathing ionic strength (0.015 M, 0.15 M, 0.5 M, 1 M, and 2 M NaCl) at room temperature. The osmotic pressure (pi) was found to increase nonlinearly with increasing chondroitin sulfate concentration and decreasing NaCl ionic bath environment. Above 1 M NaCl, pi changes negligibly with further increases in salt concentration, suggesting that Donnan osmotic pressure is negligible above this threshold, and the resulting pressure is attributed to configurational entropy. Results of the current study were also used to estimate the contribution of osmotic pressure to the stiffness of cartilage based on theoretical and experimental considerations. Our findings indicate that the osmotic pressure resulting from configurational entropy is much smaller in cartilage (based on an earlier study on bovine articular cartilage) than in free solution. The rate of change of osmotic pressure with compressive strain is found to contribute approximately one-third of the compressive modulus (H(A)(eff)) of cartilage (Pi approximately H(A)(eff)/3), with the balance contributed by the intrinsic structural modulus of the solid matrix (i.e., H(A) approximately 2H(A)(eff)/3). A strong dependence of this intrinsic modulus on salt concentration was found; therefore, it appears that proteoglycans contribute structurally to the magnitude of H(A), in a manner independent of osmotic pressure.     

Profiling DNA methylation by melting analysis

The idea of modifying DNA with bisulfite has paved the way for a variety of polymerase chain reaction (PCR) methods for accurately mapping 5-methylcytosine at specific genes. Bisulfite selectively deaminates cytosine to uracil under conditions where 5-methylcytosine remains unreacted. Following conventional PCR amplification of bisulfite-treated DNA, original cytosines appear as thymine while 5-methylcytosines appear as cytosine. Because the relative thermostability of a DNA duplex increases with increasing content of G:C base pairs, PCR products originating from DNA templates with different contents of 5-methylcytosine differ in melting temperature, i.e., the temperature required to convert the double helix into random coils. We describe two methods that resolve differentially methylated DNA sequences on the basis of differences in melting temperature. The first method integrates PCR amplification of bisulfite-treated DNA and subsequent melting analysis by using a thermal cycler coupled with a fluorometer. By including in the reaction a PCR-compatible, fluorescent dye that specifically binds to double-stranded DNA, the melting properties of the PCR product can be examined directly in the PCR tube by continuous fluorescence monitoring during a temperature transition. The second method relies on resolution of alleles with different 5-methylcytosine contents by analysis of PCR products in a polyacrylamide gel containing a gradient of chemical denaturants. Optimal resolution of differences in melting temperature is achieved by a special design of PCR primers. Both methods allow resolution of "heterogeneous" methylation, i.e., the situation where the content and distribution of 5-methylcytosine in a target gene differ between different molecules in the same sample.