Melting Curve Data Research Articles

Locked Nucleic Acids Can Enhance the Analytical Performance of Quantitative Methylation-Specific Polymerase Chain Reaction

Aberrant DNA methylation of tumor suppressor genes is a frequent epigenetic event that occurs early in tumor progression. Real-time quantitative methylation-specific polymerase chain reaction (QMSP) assays can provide accurate detection and quantitation of methylated alleles that may be potentially useful in diagnosis and risk assessment for cancer. Development of QMSP requires optimization to maximize analytical specificity and sensitivity for the detection of methylated alleles. However, in some cases challenges encountered in primer and probe design can make optimization difficult and limit assay performance. Locked nucleic acids (LNAs) demonstrate increased affinity and specificity for their cognate DNA sequences. In this proof-of-principle study, LNA residues were incorporated into primer and probe design to determine whether LNA-modified oligonucleotides could enhance the analytical performance of QMSP for IGSF4 promoter methylation in human cancer cell lines using either SYBR Green or fluorogenic probe detection methods. Use of LNA primers in QMSP with SYBR Green improved analytical specificity for methylated alleles and eliminated the formation of nonspecific products because of mispriming from unmethylated alleles. QMSP using LNA probe and primers showed an increased amplification efficiency and maximum fluorescent signal. QMSP with LNA oligonucleotides and either detection method could reliably detect five genome equivalents of methylated DNA in 1000- to 10,000-fold excess unmethylated DNA. Thus, LNA oligonucleotides can be used in QMSP optimization to enhance analytical performance.

Role of Nicotinic and Estrogen Signaling during Experimental Acute and Chronic Bladder Inflammation

Inflammation is a physiological process that characterizes many bladder diseases. We hypothesized that nicotinic and estrogen signaling could down-regulate bladder inflammation. Cyclophosphamide was used to induce acute and chronic bladder inflammation. Changes in bladder inflammation were measured histologically and by inflammatory gene expression. Antagonizing nicotinic signaling with mecamylamine further aggravated acute and chronic inflammatory changes resulting from cyclophosphamide treatment. Estrogen and nicotinic signaling independently attenuated acute bladder inflammation by decreasing neutrophil recruitment and down-regulating elevated lipocalin-2 and cathepsin D expression. However, the combined signaling by the estrogen and nicotinic pathways, as measured by macrophage infiltration and up-regulation of interleukin-6 expression in the bladder, synergistically reduced chronic bladder inflammation. The elevated expression of p65 nuclear localization in bladders treated with cyclophosphamide or cyclophosphamide with mecamylamine suggested nuclear factor-kappa B activation in the chronic inflammatory process. The complementary treatment of 17 beta-estradiol and the nicotinic agonist anabasine resulted in the translocation of p65 to the cytoplasm, again greater than either alone. Activation of nuclear factor-kappaB can result in macrophage activation and/or elevation in epithelial proliferation. These data suggest that 17 beta-estradiol and anabasine reduce chronic bladder inflammation through reduction of nuclear translocation of p65 to suppress cytokine expression.

High-Throughput SNP Genotyping by Single-Tube PCR with T m -Shift Primers

Despite many recent advances in high-throughput single nucleotide polymorphism (SNP) genotyping technologies, there is still a great need for inexpensive and flexible methods with a reasonable throughput. Here we report substantial modifications and improvements to an existing homogenous allele-specific PCR-based SNP genotyping method, making it an attractive new option for researchers engaging in candidate gene studies or following up on genome-wide scans. In this advanced version of the melting temperature (Tm)-shift SNP genotyping method, we attach two GC-rich tails of different lengths to allele-specific PCR primers, such that SNP alleles in genomic DNA samples can be discriminated by the Tms of the PCR products. We have validated 306 SNP assays using this method and achieved a success rate in assay development of greater than 83% under uniform PCR conditions. We have developed a standalone software application to automatically assign genotypes directly from melting curve data. To demonstrate the accuracy of this method, we typed 592 individuals for 6 SNPs and showed a high call rate (>98%) and high accuracy (>99.9%). With this method, 6-10,000 samples can be genotyped per day using a single 384-well real-time thermal cycler with 2-4 standard 384-well PCR instruments.

Detection of Shiga toxin genes stx1, stx2, and the +93 uidA mutation of E. coli O157:H7/H-using SYBR® Green I in a real-time multiplex PCR

Enterohemorrhagic Escherichia coli (EHEC) is a major foodborne pathogen capable of causing diarrhea and vomiting, but more serious complications such as hemorrhagic colitis and hemolytic-uremic syndrome (HUS) can result. A real-time PCR method to detect the presence of Shiga toxin producing E. coli (STEC) and E. coli O157:H7 was investigated using SYBR Green I (SG). Primers were designed to target the Shiga toxin genes (stx1 and stx2) and a highly conserved base substitution at +93 of the beta-glucuronidase gene (uidA) unique to E. coli O157:H7. An initial test panel of five E. coli and non-E. coli isolates was tested with individual primer sets (simplex assay) and all primer sets including stx1, stx2, and uidA (multiplex assay). All strains were correctly identified in both assays. Average melt temperatures (Tm's, degrees C) for PCR products were 85.42--stx1, 81.93--stx2, and 88.25--uidA in simplex assays and 85.20--stx1, 81.20--stx2, and 88.16--uidA when multiplexed. Each of the three gene targets in one multiplex reaction could be distinguished by melt curve data with significantly different Tm's. The assay was expanded to a panel of 138 isolates consisting of STEC, E. coli O157:H7, non-toxigenic E. coli, and non-E. coli isolates with melt peaks consistent with those stated above.

Recognition of CG inversions in DNA triple helices by methylated 3H-pyrrolo[2,3-d]pyrimidin-2(7H)-one nucleoside analogues

Substituted 3H-pyrrolo[2,3-d]pyrimidin-2(7H)-one nucleoside analogues have been synthesised from 5-alkynyl-uridine derivatives, incorporated into triplex forming oligonucleotides (TFOs) and found to selectively bind CG inversions with enhanced affinity compared to T.

Ferrocenyl‐Modified DNA: Synthesis, Characterization and Integration with Semiconductor Electrodes

The ferrocenyl-nucleoside, 5-ethynylferrocenyl-2'-deoxycytidine (1) has been prepared by Pd-catalyzed cross-coupling between ethynylferrocene and 5-iodo-2'-deoxycytidine and incorporated into oligonucleotides by using automated solid-phase synthesis at both silica supports (CPG) and modified single-crystal silicon electrodes. Analysis of DNA oligonucleotides prepared and cleaved from conventional solid supports confirms that the ferrocenyl-nucleoside remains intact during synthesis and deprotection and that the resulting strands may be oxidised and reduced in a chemically reversible manner. Melting curve data show that the ferrocenyl-modified oligonucleotides form duplex structures with native complementary strands. The redox potential of fully solvated ferrocenyl 12-mers, 350 mV versus SCE, was shifted by +40 mV to a more positive potential upon treatment with the complement contrary to the anticipated negative shift based on a simple electrostatic basis. Automated solid-phase methods were also used to synthesise 12-mer ferrocenyl-containing oligonucleotides directly at chemically modified silicon <111> electrodes. Hybridisation to the surface-bound ferrocenyl-DNA caused a shift in the reduction potential of +34 mV to more positive values, indicating that, even when a ferrocenyl nucleoside is contained in a film, the increased density of anions from the phosphate backbone of the complement is still dominated by other factors, for example, the hydrophobic environment of the ferrocene moiety in the duplex or changes in the ferrocene-phosphate distances. The reduction potential is shifted >100 mV after hybridisation when the aqueous electrolyte is replaced by THF/LiClO(4), a solvent of much lower dielectric constant; this is consistent with an explanation based on conformation-induced changes in ferrocene-phosphate distances.

Validation of an algorithm for automatic quantification of nucleic acid copy numbers by real-time polymerase chain reaction

Real-time quantitative polymerase chain reaction (PCR) with on-line fluorescence detection has become an important technique not only for determination of the absolute or relative copy number of nucleic acids but also for mutation detection, which is usually done by measuring melting curves. Optimum assay conditions have been established for a variety of targets and experimental setups, but only limited attention has been directed to data evaluation and validation of the results. In this work, algorithms for the processing of real-time PCR data are evaluated for several target sequences ( p53, IGF-1, PAI-1, Factor VIIc) and compared to the results obtained by standard procedures. The algorithms are implemented in software called SoFAR, which allows fully automatic analysis of real-time PCR data obtained with a Roche LightCycler instrument. The software yields results with considerably increased precision and accuracy of quantifications. This is achieved mainly by the correction of amplification-independent signal trends and a robust fit of the exponential phase of the signal curves. The melting curve data are corrected for signal changes not due to the melting process and are smoothed by fitting cubic splines. Therefore, sensitivity, resolution, and accuracy of melting curve analyses are improved.

Melting curve equations at high pressure

A number of equations for the melting temperature dependence on pressure, ${T}_{m}(P),$ based on the two- and one-phase approaches to melting have been obtained. All melting curves have the form ${T}_{m}=F(P)D(P),$ where $F(P)$ is the Simon (rising) melting equation and $D(P)$ is the damping function, which asymptotically slopes down under pressure. This form predicts that each solid phase has a maximum melting temperature at positive or negative pressure. The simplest equation of this form is ${T}_{m}{=T}_{0}(1+\ensuremath{\Delta}{P/a)}^{b}\mathrm{exp}(\ensuremath{-}c\ensuremath{\Delta}P),$ where $\ensuremath{\Delta}P=P\ensuremath{-}{P}_{0},$ ${P}_{0}$ is some reference pressure, and a, b, and c are parameter which are identified in terms of thermodynamic values. All melting curve data obey this equation. This implies that there exist no anomalous melting curves. All melting curves, rising, falling, and flattening, as well as curves with a maximum, are normal insofar as all of them can be described by the unified equation.

Elastic properties of substances in the megabar pressure range: Inversion of shear rigidity

The behavior of elastic moduli of substances is analyzed in the megabar pressure range. A new effect—inversion of the shear moduli and mechanical properties upon compression—is predicted for various classes of substances. The melting-curve data for different materials confirm the predicted phenomenon. The materials traditionally considered the softest, such as rare gas solids and molecular substances, may become the hardest in the megabar range. This should be taken into account in developing the experimental high-pressure technique.

The Parallel and Antiparallel Triplex Formation and Stability of Self Complementary Oligonucleotides Containing 2′-Fluoro-arabinosyl Thymine and 5-Methyl-2′-Deoxycytidine

We examined the effects of 1–(2-deoxy -2-fluoro-β-D-arabinofuranosyl)-thymine (or FMAU, a potent antiviral nucleoside) on the stability of duplex and triplexes. When compared the stability of the self-complementary 5′-A5T5 duplex with 5′-A5X5 (X = FMAU), duplex containing FMAU has much higher melting temperature (Tm). 5′-A6T5T3X3T5F3X3 and T3X3T5A6T5F3X3 form the parallel and antiparallel triplexes T3X3: A6:X3X3, respectively. The former exhibited the typical T:A:T triplex behavior with only one melting temperature at 70 °C and 45 °c in 1.0 M and 0.2 M NaCl solution, respectively, whereas the latter has two Tm values at 56 °C and 28 °C in 1.0 M solution. FMAU clearly stabilize the triplex structure as A6T22 which forms the parallel triplex T6:A6:T6 has also only one Tm at 54 °C and 37 °C in high and iow salt concentration solutions, respectively. A 31mer 5′-TCCTCCTTTTTTAGGAGGATTTTTTGGTGGT and 5′-TCCTCCTTTTTTAGGAGGATTTTTTX'X'TX'X'T (X' = 2′-deoxy-5-methylcytidine) were prepared to study their triplex forming potential. The former was found to have a week interaction of the Watson-Crick duplex with the mismatched third-strand at all pH. The latter formed a stable triplex at lower pH consistent with required protonation on the 5-methylcytosine base. For these studies we developed a simple PC desktop spreadsheet program to calculate the first derivative profile of the melting curve data. This paper is dedicated to Prof. Jacques H. van Boom on the occasion of his 60th birthday.

Vacancies in solid4He: Conflicting experimental evidence

We compare the properties of vacancies in4He implied by X-ray, NMR, and acoustic measurements with specific heat, pressure, and melting curve data. Taken together, these different measurements indicate that the model of vacancies as occupying a narrow band of states cannot be correct. Better agreement can be obtained by assuming that vacancies occupy a wide band but inconsistencies remain. An unambiguous determination of the properties of vacancies requires additional experimental and theoretical work.

Melting of forsterite, Mg2SiO4, from 9.7 to 16.5 GPa

A multianvil apparatus has been used to determine the pressure‐temperature melting curve of forsterite from 9.7 to 16.5 GPa. At 10.1 GPa a singular point occurs that marks the change from congruent melting at lower pressures to incongruent melting (forsterite = periclase + liquid) at higher pressures. The melting curve also passes through two invariant points. At one (15.6 GPa, 2310°C), the phases forsterite, periclase, anhydrous B, and liquid coexist and the melting reaction changes from forsterite = periclase + liquid at lower pressures to forsterite = anhydrous B + liquid at higher pressures. At the other (16.7 GPa, 2315°C), the phases forsterite, modified spinel, anhydrous B, and liquid coexist, and the melting reaction changes from forsterite = anhydrous B + liquid at lower pressures to modified spinel = anhydrous B + liquid at higher pressures. The Simon equation, P(GPa) = 2.44[(T(°K)/2171)11.4 − 1], fits both our melting curve data and the lower pressure data of Davis and England (1964). At low pressures, the melting curve of forsterite lies at higher temperatures than that of enstatite, but the two curves cross at 13.3 GPa because of the lower dT/dP slope of the forsterite melting curve. This causes the forsterite‐enstatite eutectic to shift toward forsterite as pressure increases, but our data are consistent with earlier findings that the shift is not sufficient to support an origin for the mantle by eutectic‐like melting at high pressures.

Equation of state of sodium

The volume of sodium in the bcc structure was measured at 293 K to 9 GPa using a high pressure, x-ray diffraction technique. The compression of NaF was used as the pressure gauge. These data, the shock compression data of Rice and Bakanova et al., and the melting curve data of Luedemann and Kennedy, and Ivanov et al., are all used to establish a model for the equation of state of sodium.

Influence of base-pair changes and cooperativity parameters on the melting curves of short DNAs.

AbstractTheoretical melting curves were calculated for four DNA restriction fragments, 157–257 base pairs (bp), and a series of hypothetical block DNAs with sequences d(C2xAxC2x). d(C2xTxG2x), 5 ⩽ x ⩽ 40. These DNAs provided a mixture of A·T/G·C sequence distributions with which to investigate the effects of parameters and base‐pair changes on the melting of short DNAs. The sensitivity of DNA melting curves to changes in internal loop melting parameters σ and κ was examined. As Expected, theoretical melting curves of short DNAs with a quasirandom base‐pair sequence vary little with changes in internal loop parameters. End melting dominates the transition behaviour of these moleucles. This was also observed for the block DNAs up to x = 22. Beyond this length, melting curves are highly sensitive to the internal loop parameters. Sensitivity is also predicted for a 157‐bp fragment with a block distribution of A·T and G·C pairs. These results indicate that accurate evaluation of internal loop parameters is possible with short DNAs (100–200 bp) containing a G·C/A·T/G·C block distribution with at least 22 bp in each block. Duplex‐to‐single‐strands dissociation parameters were reevaluated form experimental melting curve data of eight DNA fragments using a least squares fit approach. This analysis confirmed parameter values previously found with a simplified dissociation model. A Priori predictions are made on the effects of base‐pair changes on the melting curves of three characterized DNA restriction fragments. Single base‐pair changes are predicted to induce small but measurable changes in the melting curves. The characteristics of the altered melting curves depend on the location of the base‐pair change.

Liquid alkali metals and alloys as electron-ion plasmas

Linear screening theory yields expressions for the structure of liquid metals and alloys in terms of the structure of a bare ionic plasma on a rigid background, of the dielectric function of the homogeneous electron gas, and of electron-ion pseudopotentials. The results of such an electron-ion plasma model are examined against experimental data on the structural and thermodynamic properties of the liquid alkali metals and their alloys. The calculations rely on a very accurate theoretical determination of the structure factor of the classical ionic plasma, which embodies its known equation of state and thermodynamic consistency. The model gives a rather accurate account ofthe compressibility of the liquid metal. The main empirical adjustments that are included at finite wavenumber are a choice of the plasma parameter at freezing and a large momentum cutoff of the electron-ion coupling, as suggested by melting-curve data and by a discussion of dynamic criteria for the solid-liquid transition in the alkali metals. The model is then shown to yield very good agreement with experiment for the detailed shape of the structure factor of the liquid metal near freezing, and for its dependence on pressure, temperature and density. A discrepancy with experiment is found in the description of long-wavelength concentration fluctuations in alloys and is attributed to electronic charge transfer between the constituents, which is not included in the model. An estimation of the spinodal curve for the Li-Na alloy is presented in support of this conjecture.

Zero sound and the melting curve of 3He

We have analyzed data from existing accurate pressure and molar volume measurements of 3He along the melting curve to determine explicit entropy contributions of the liquid and solid phases. Below 300 mK there is a significant discrepancy between the melting curve data of Grilly 1 and Halperin, et al . 2 The resolution of this problem is the subject of this paper. We find that zero sound makes an important contribution to the liquid entropy and that the superfluid 3He A-transition temperature must be shifted upward.

Effective mass of liquid3He using the melting curve data

Measurements of dp/dt and of the liquid and solid molar volumes along the melting curve of 3He can be used to obtain information about the Fermi liquid parameter Ef=pf2/2m* of liquid 3He. The authors use data at temperatures reasonably far above the Neel temperature of the solid, but low enough so that the first finite-temperature correction term in Fermi liquid theory is not large. It is shown that, in spite of uncertainties in the solid 3He entropy because of uncertainties in the knowledge of the exchange mechanism, the melting curve data provide a check on the different sets of values for m* quoted in literature. The authors also discuss the possible effect of ground state vacancies, whose existence in BCC 3He has been speculated on this analysis.

Conformational studies on copolymers of L-lysine and L-leucine: circular dichroism and potentiometric titration studies.

AbstractConformational aspects of a series of copolymers of L‐Leucine and L‐leucine [poly‐(LysxLeuy)] containing 0 to 0.41 mole fraction L‐leucine have been studied by circular dichroism (CD) and potentiometric titration in 0.05M KF solution. CD studies on the α‐helical conformation showed a dependence of the magnitude of the CD ellipticity band at 222 nm on copolymer composition; the [θ]222 decreasing with higher leucine contents. This was interpreted as the result of an increase of the hydrophobicity of the environment of the amide group due to the presence of the leucyl residues. Values of the Zimm‐Rice parameter, σ, for the copolymers were obtained from the potentiometric titrations and used to fit theoretical curves to the experimental data. Using the variation of σ with polymer composition, a value of σ for the leucyl residue was estimated to be 6.3 × 10−2, assuming independence of σ on the amino acid sequence in the copolymer. The free energy change for the conversion of one mole residue from uncharged helix to uncharged coil, ΔGhc°, was also obtained from the titration data for each copolymer up to a leucine mole fraction of 0.16; a value of 385 cal mole−1 was estimated for ΔGhc° for a leucyl residue. These values for σ and ΔGhc° are compared with other values in the literature for various amino acid residues obtained from titration and melting curve data.

Arrangement of the highly reiterated DNA sequences in the centric heterochromatin of Drosophila melanogaster evidence for interspersed spacer DNA

We have investigated the arrangement of the highly reiterated centromeric DNA sequences in Drosophila melanogaster by studying the physical properties of sequences obtained after the hydroxyapatite isolation of rapidly renaturing molecules. It was found that the fraction of material recovered as partially renatured varied with the molecular weight of the starting material, although conservative length corrections on the chosen C 0t values were made. On banding in a CsCl gradient a bimodal distribution for the renatured molecules was found. This bimodal distribution was also sensitive to the molecular weight of the single strand material. The peaks were shown to consist of respectively: h.a.r.r.DNA † † Abbreviation used: h.a.r.r.DNA; hydroxy- apatite isolated rapidly renaturing DNA. “Pure h.a.r.r.DNA” refers to h.a.r.r.DNA isolated from extensively sheared DNA (mol. wt. 200,000), in order to free the highly reiterated sequences from attachment to less redundant or “unique” DNA. sequences and renatured h.a.r.r.DNA sequences with attached unrenatured “spacer” sequences. From melting curve data, RNA-DNA hybridization and kinetic analysis other explanations for the increased yield and bimodal distribution of the renatured molecules could be ruled out. Computer simulation of a random shear process on a DNA sequence which alternates an h.a.r.r.DNA sequence from one to three times larger than the spacer sequence can be seen to fit the experimental situation. We conclude that the constitutive heterochromatin is composed of blocks of extremely simple sequences stretching from 1.5 to 6 million daltons interspersed by a more complex sequence of molecular weights of approximately 2 to 4 million daltons. A similar interspersion of sequences for the mouse satellite DNA was found, though preliminary data suggest that the stretches of mouse satellite may be much longer. The occurrence of a more complex DNA at this locus of the Drosophila chromosome raises some interesting possibilities for both the genetic potential of this area and for chromosomal “housekeeping” functions.

Experimental Determination of the P—T Melting Curves of Kr, Ne, and He

Pressure—temperature melting data have been obtained at pressures of 0–8 kbar for Kr, 0–10 kbar for Ne, and 1–10 kbar for He. The estimated accuracy of the data is ± 1 bar and ± 0.002 to ± 0.011oK. Use of a modified Simon melting equation of the form P=A(T+D)c+B to represent the data removes nearly all of the systematic deviations which are observed when the usual Simon melting equation P=ATc+B is applied. The modified form of the equation also provides much better high pressure extrapolations from the melting curve data.