Enhancements and modifications of primer design program Primer3

The determination of annealing temperature is a critical step in PCR design. This parameter is typically derived from the melting temperature of the PCR primers, so for successful PCR work it is important to determine the melting temperature of primer accurately. We introduced several enhancements in the widely used primer design program Primer3. The improvements include a formula for calculating melting temperature and a salt correction formula. Also, the new version can take into account the effects of divalent cations, which are included in most PCR buffers. Another modification enables using lowercase masked template sequences for primer design. Availability: Features described in this article have been implemented into the development code of Primer3 and will be available in future versions (version 1.1 and newer) of Primer3. Also, a modified version is compiled under the name of mPrimer3 which is distributed independently. The web-based version of mPrimer3 is available at http://bioinfo.ebc.ee/mprimer3/ and the binary code is freely downloadable from the URL http://bioinfo.ebc.ee/download/.     

两价盐对C_8－卵磷脂微团溶液液－液相分离的影响

采用激光散射等方法测定了在添加不同的两价无机盐情况下,Ｃ８－卵磷脂微团溶液的液－液相分离曲线,及其相变临界温度随盐类型和盐离子强度的变化。并从理论上分析了两价盐对Ｃ８－卵磷脂微团溶液吉布斯自由能的影响,推导出－关于盐对该微团溶液相交临界温度影响的半经验半理论公式,可满意地描述该微团溶液的液－液相分离受益调控的规律。     

两价盐对C_8－卵磷脂微团溶液液－液相分离的影响

采用激光散射等方法测定了在添加不同的两价无机盐情况下,Ｃ８－卵磷脂微团溶液的液－液相分离曲线,及上变临界温度随盐类型和盐离子强度的变化,并从理论上分析两价盐对Ｃ８－卵磷脂微团溶液吉布斯自由能的影响,推导出－关于盐对该微团溶液相变临界温度影响的半经验半理论公式,可满意地描述该微团溶液的液－液相分离受盐调控的规律。     

Universal primers for fluorescent labelling of PCR fragments--an efficient and cost-effective approach to genotyping by fluorescence

Directly labelling locus‐specific primers for microsatellite analysis is expensive and a common limitation to small‐budget molecular ecology projects. More cost‐effective end‐labelling of PCR products can be achieved through a three primer PCR approach, involving a fluorescently labelled universal primer in combination with modified locus‐specific primers with 5′ universal primer sequence tails. This technique has been widely used but has been limited largely due to a lack of available universal primers suitable for co‐amplifying large numbers of size overlapping loci and without requiring locus‐specific PCR conditions to be modified. In this study, we report a suite of four high‐performance universal primers that can be employed in a three primer PCR approach for efficient and cost‐effective fluorescent end‐labelling of PCR fragments. Amplification efficiency is maximized owing to high universal primer Tm values (approximately 60+ °C) that enhance primer versatility and enable higher annealing temperatures to be employed compared with commonly used universal primers such as M13. We demonstrate that these universal primers can be combined with multiple fluorophores to co‐amplify multiple loci efficiently via multiplex PCR. This method provides a level of multiplexing and PCR efficiency similar to microsatellite fluorescent detection assays using directly labelled primers while dramatically reducing project costs. Primer performance is tested using several alternative PCR strategies that involve both single and multiple fluorophores in single and multiplex PCR across a wide range of taxa.     

Predicting stability of DNA duplexes in solutions containing magnesium and monovalent cations

Accurate predictions of DNA stability in physiological and enzyme buffers are important for the design of many biological and biochemical assays. We therefore investigated the effects of magnesium, potassium, sodium, Tris ions, and deoxynucleoside triphosphates on melting profiles of duplex DNA oligomers and collected large melting data sets. An empirical correction function was developed that predicts melting temperatures, transition enthalpies, entropies, and free energies in buffers containing magnesium and monovalent cations. The new correction function significantly improves the accuracy of predictions and accounts for ion concentration, G-C base pair content, and length of the oligonucleotides. The competitive effects of potassium and magnesium ions were characterized. If the concentration ratio of [Mg (2+)] (0.5)/[Mon (+)] is less than 0.22 M (-1/2), monovalent ions (K (+), Na (+)) are dominant. Effects of magnesium ions dominate and determine duplex stability at higher ratios. Typical reaction conditions for PCR and DNA sequencing (1.5-5 mM magnesium and 20-100 mM monovalent cations) fall within this range. Conditions were identified where monovalent and divalent cations compete and their stability effects are more complex. When duplexes denature, some of the Mg (2+) ions associated with the DNA are released. The number of released magnesium ions per phosphate charge is sequence dependent and decreases surprisingly with increasing oligonucleotide length.     

Mg2+和Na+对多核酶系统体外切割反应的影响

为了进一步了解2价Mg2+和1价Na+存在与否的情况下,多核酶系统对底物RNA的切割效率,构建了pGEM-Coat'A,pGEM-Coat'A196Rz质粒和pGEM-MDR1靶质粒,通过用SP6/T7转录试剂盒在体外转录RNA,在无细胞系统进行切割反应,反应产物通过6%变性聚丙烯酰胺凝胶电泳,干胶、x光片曝光自显影,利用Image J生物图像分析软件分析,结果表明,多核酶系统的切割效率依赖于二价Mg2+的浓度,切割产物随Mg2+浓度的增加而增加,而且具有反应时间的依赖性,在Na+浓度低于200 mmol/L且单独存在时,没有切割产物生成,相反,在Na+和Mg2+共存时,表现出Na+抑制Mg2+诱导的切割活性,切割效率明显低于Mg2+单独存在时的结果.这些结果提示,在生理环境下,Mg2+对于多核酶系统对底物的切割反应是必需的,而Na+则不是.     

DNA like-charge attraction and overcharging by divalent counterions in the presence of divalent co-ions

Strongly correlated electrostatics of DNA systems has drawn the interest of many groups, especially the condensation and overcharging of DNA by multivalent counterions. By adding counterions of different valencies and shapes, one can enhance or reduce DNA overcharging. In this paper, we focus on the effect of multivalent co-ions, specifically divalent co-ions such as SO\(_{4}^{2-}\). A computational experiment of DNA condensation using Monte Carlo simulation in grand canonical ensemble is carried out where the DNA system is in equilibrium with a bulk solution containing a mixture of salt of different valency of co-ions. Compared to systems with purely monovalent co-ions, the influence of divalent co-ions shows up in multiple aspects. Divalent co-ions lead to an increase of monovalent salt in the DNA condensate. Because monovalent salts mostly participate in linear screening of electrostatic interactions in the system, more monovalent salt molecules enter the condensate leads to screening out of short-range DNA–DNA like charge attraction and weaker DNA condensation free energy. The overcharging of DNA by multivalent counterions is also reduced in the presence of divalent co-ions. Strong repulsions between DNA and divalent co-ions and among divalent co-ions themselves lead to a depletion of negative ions near the DNA surface as compared to the case without divalent co-ions. At large distances, the DNA–DNA repulsive interaction is stronger in the presence of divalent co-ions, suggesting that divalent co-ions’ role is not only that of simple stronger linear screening.     

MethPrimer: designing primers for methylation PCRs

MOTIVATION: DNA methylation is an epigenetic mechanism of gene regulation. Bisulfite- conversion-based PCR methods, such as bisulfite sequencing PCR (BSP) and methylation specific PCR (MSP), remain the most commonly used techniques for methylation mapping. Existing primer design programs developed for standard PCR cannot handle primer design for bisulfite-conversion-based PCRs due to changes in DNA sequence context caused by bisulfite treatment and many special constraints both on the primers and the region to be amplified for such experiments. Therefore, the present study was designed to develop a program for such applications. RESULTS: MethPrimer, based on Primer 3, is a program for designing PCR primers for methylation mapping. It first takes a DNA sequence as its input and searches the sequence for potential CpG islands. Primers are then picked around the predicted CpG islands or around regions specified by users. MethPrimer can design primers for BSP and MSP. Results of primer selection are delivered through a web browser in text and in graphic view.     

RNA helix stability in mixed Na+/Mg2+ solution

A recently developed tightly bound ion model can account for the correlation and fluctuation (i.e., different binding modes) of bound ions. However, the model cannot treat mixed ion solutions, which are physiologically relevant and biologically significant, and the model was based on B-DNA helices and thus cannot directly treat RNA helices. In the present study, we investigate the effects of ion correlation and fluctuation on the thermodynamic stability of finite length RNA helices immersed in a mixed solution of monovalent and divalent ions. Experimental comparisons demonstrate that the model gives improved predictions over the Poisson-Boltzmann theory, which has been found to underestimate the roles of multivalent ions such as Mg2+ in stabilizing DNA and RNA helices. The tightly bound ion model makes quantitative predictions on how the Na+-Mg2+ competition determines helix stability and its helix length-dependence. In addition, the model gives empirical formulas for the thermodynamic parameters as functions of Na+/Mg2+ concentrations and helix length. Such formulas can be quite useful for practical applications.     

Thermodynamic parameters for the binding of divalent cations to gramicidin A incorporated into a lipid environment by Tl-205 nuclear magnetic resonance.

Thermodynamic parameters, enthalpy and entropy, for the binding of the divalent cations, Mg+2, Ca+2, Sr+2, Ba+2, and Cd+2, to gramicidin A, incorporated into lysophosphatidylcholine, have been determined using a combination of Tl-205 nuclear magnetic resonance spectroscopy and competition binding. The binding process is thermodynamically driven by the enthalpy and not the entropy. The enthalpy values are related to the process involving the transfer of cations from an aqueous environment to an amide environment. A comparison is made between the thermodynamic parameters for the binding of monovalent and divalent cations to gramicidin A to illustrate the channel blocking ability of the divalent cations with respect to monovalent cation transport.     

Structural characterization of a ribonuclease III processing signal.

The structure of a ribonuclease III processing signal from bacteriophage T7 was examined by NMR spectroscopy, optical melting, and chemical and enzymatic modification. A 41 nucleotide variant of the T7 R1.1 processing signal has two Watson-Crick base-paired helices separated by an internal loop, consistent with its predicted secondary structure. The internal loop is neither rigidly structured nor completely exposed to solvent, and seems to be helical. The secondary structure of R1.1 RNA is largely insensitive to the monovalent cation concentration, which suggests that the monovalent cation sensitivity of secondary site cleavage by RNase III is not due to a low salt-induced RNA conformational change. However, spectroscopic data show that Mg2+ affects the conformation of the internal loop, suggesting a divalent cation binding site(s) within this region. The Mg(2+)-dependence of RNase III processing of some substrates may reflect not only a requirement for a divalent cation as a catalytic cofactor, but also a requirement for a local RNA conformation which is divalent cation-stabilized.     

An improved method for production of silica from rice hull ash

Biosorption of monovalent ions Na+ and K+, by deactivated protonated yeast (Saccharomyces cerevisiae) at controlled pH, was compared with biosorption of divalent ions Ca2+ and Mg2+ to help to understand the underlying bindingmechanisms. The adsorption for monovalent ions was accompanied by H+ release. Divalent ions were sorbed by proton displacement, and also an additional mode not accompanied by release of H+. The sorption uptake of both monovalent and divalent metal ions increased with pH in the range 3-7 peaking at 6.75. Equilibrium sorption isotherms at pH = 6.75 showed that the totalmaximum biosorptive capacity for metal ions decreased in the following order: Ca > Mg > Na > or = K.     

Permeation of monovalent cations through the non-capacitative arachidonate-regulated Ca2+ channels in HEK293 cells. Comparison with endogenous store-operated channels

In a manner similar to voltage-gated Ca(2+) channels and Ca(2+) release-activated Ca(2+) (CRAC) channels, the recently identified arachidonate-regulated Ca(2+) (ARC) channels display a large monovalent conductance upon removal of external divalent cations. Using whole-cell patch-clamp recording, we have characterized the properties of these monovalent currents in HEK293 cells stably transfected with the m3 muscarinic receptor and compared them with the corresponding currents through the endogenous store-operated Ca(2+) (SOC) channels in the same cells. Although the monovalent currents seen through these two channels displayed certain similarities, several marked differences were also apparent, including the magnitude of the monovalent current/Ca(2+) current ratio, the rate and nature of the spontaneous decline in the currents, and the effects of external monovalent cation substitutions and removal of internal Mg(2+). Moreover, monovalent ARC currents could be activated after the complete spontaneous inactivation of the corresponding SOC current in the same cell. We conclude that the non-capacitative ARC channels share, with voltage-gated Ca(2+) channels and store-operated Ca(2+) channels (e.g. SOC and CRAC the general property of monovalent ion permeation in the nominal absence of extracellular divalent ions. However, the clear differences between the properties of these currents through ARC and SOC channels in the same cell confirm that these represent distinct conductances.     

Electron and scanning force microscopy studies of alterations in supercoiled DNA tertiary structure.

The configuration of supercoiled DNA (scDNA) was investigated by electron microscopy and scanning force microscopy. Changes in configuration were induced by varying monovalent/divalent salt concentrations and manifested by variation in the number of nodes (crossings of double helical segments). A decrease in the concentration of monovalent cations from 50 mM to approximately 1 mM resulted in a significant change of apparent configuration of negatively supercoiled DNA from a plectonemic form with virtually approximately 15 nodes (the value expected for molecules of approximately 3000 bp) to one or two nodes. This result was in good agreement with values calculated using an elastic rod model of DNA and salt concentration in the range of 5-50 mM. The effect did not depend on the identity of the monovalent cation (Na(+), K(+)) or the nature of the support used for electron microscopy imaging (glow-discharged carbon film, polylysine film). At very low salt concentrations, a single denatured region several hundred base-pairs in length was often detected. Similarly, at low concentrations of divalent cations (Mg(2+), Ca(2+), Zn(2+)), scDNA was apparently relaxed, although the effect was slightly dependent on the nature of the cation. Positively supercoiled DNA behaved in a manner different from that of its negative counterpart when the ion concentration was varied. As expected for these molecules, an increase in salt concentration resulted in an apparent relaxation; however, a decrease in salt concentration also led to an apparent relaxation manifested by a slight decrease in the number of nodes. Scanning force microscopy imaging of negatively scDNA molecules deposited onto a mica surface under various salt conditions also revealed an apparent relaxation of scDNA molecules. However, due to weak interactions with the mica surface in the presence of a mixture of mono/divalent cations, the effect occurred under conditions differing from those used for electron microscopy. We conclude that the observed changes in scDNA configuration are inherent to the DNA structure and do not reflect artifacts arising from the method(s) of sample preparation.     

Limiting-laws of polyelectrolyte solutions. Ionic distribution in mixed-valency counterions systems. II. A comparison of conductometric data and theoretical predictions

The competitive binding of monovalent and divalent counterions (M+ and M2+, respectively) has been studied by a conductometric procedure as described by De Jong et al. (Biophysical Chemistry 27 (1987) 173) for aqueous solutions of alkali metal polymethacrylates in the presence of Ca (NO3)2 or Mg(NO3)2. The experimentally obtained fractions of conductometrically free counterions are compared with theoretical values computed according to a new thermodynamic model recently developed by Paoletti et al. (Biophysical Chemistry, 41 (1991) 73). For the systems studied, the fractions of free monovalent and divalent counterions can be fairly well described by the theory. In fact, the results support the assumption that under the present conditions the conductometrically obtained distribution parameters (l) and (2) approximate the equilibrium fractions of free monovalent and divalent counterions. For a degree of neutralization of 0.8 and a molar concentration ratio of divalent counterions and charged groups on the polyion up to 0.25, the mean M+/M2+, exchange ratio nu has been found to be 1.39 +/- 0.03 and 1.33 +/- 0.03 for the alkali metal/Ca/PMA and alkali metal/Mg/PMA systems, respectively. These values agree well with the theoretical value, which for this particular case is 1.38.     

PCR detection of nearly any dengue virus strain using a highly sensitive primer 'cocktail'

PCR detection of viral pathogens is extremely useful, but suffers from the challenge of detecting the many variant strains of a given virus that arise over time. Here, we report the computational derivation and initial experimental testing of a combination of 10 PCR primers to be used in a single high-sensitivity mixed PCR reaction for the detection of dengue virus. Primer sequences were computed such that their probability of mispriming with human DNA is extremely low. A 'cocktail' of 10 primers was shown experimentally to be able to detect cDNA clones representing the four serotypes and dengue virus RNA spiked into total human whole blood RNA. Computationally, the primers are predicted to detect 95% of the 1688 dengue strains analyzed (with perfect primer match). Allowing up to one mismatch and one insertion per primer, the primer set detects 99% of strains. Primer sets from three previous studies have been compared with the present set of primers and their relative sensitivity for dengue virus is discussed. These results provide the formulation and demonstration of a mixed primer PCR reagent that may enable the detection of nearly any dengue strain irrespective of serotype, in a single PCR reaction, and illustrate an approach to the broad problem of detecting highly mutable RNA viruses.     

Non-specific binding of Na+ and Mg2+ to RNA determined by force spectroscopy methods

RNA duplex stability depends strongly on ionic conditions, and inside cells RNAs are exposed to both monovalent and multivalent ions. Despite recent advances, we do not have general methods to quantitatively account for the effects of monovalent and multivalent ions on RNA stability, and the thermodynamic parameters for secondary structure prediction have only been derived at 1M [Na(+)]. Here, by mechanically unfolding and folding a 20 bp RNA hairpin using optical tweezers, we study the RNA thermodynamics and kinetics at different monovalent and mixed monovalent/Mg(2+) salt conditions. We measure the unfolding and folding rupture forces and apply Kramers theory to extract accurate information about the hairpin free energy landscape under tension at a wide range of ionic conditions. We obtain non-specific corrections for the free energy of formation of the RNA hairpin and measure how the distance of the transition state to the folded state changes with force and ionic strength. We experimentally validate the Tightly Bound Ion model and obtain values for the persistence length of ssRNA. Finally, we test the approximate rule by which the non-specific binding affinity of divalent cations at a given concentration is equivalent to that of monovalent cations taken at 100-fold concentration for small molecular constructs.     

Distinct properties of CRAC and MIC channels in RBL cells

In rat basophilic leukemia (RBL) cells and Jurkat T cells, Ca(2+) release-activated Ca(2+) (CRAC) channels open in response to passive Ca(2+) store depletion. Inwardly rectifying CRAC channels admit monovalent cations when external divalent ions are removed. Removal of internal Mg(2+) exposes an outwardly rectifying current (Mg(2+)-inhibited cation [MIC]) that also admits monovalent cations when external divalent ions are removed. Here we demonstrate that CRAC and MIC currents are separable by ion selectivity and rectification properties: by kinetics of activation and susceptibility to run-down and by pharmacological sensitivity to external Mg(2+), spermine, and SKF-96365. Importantly, selective run-down of MIC current allowed CRAC and MIC current to be characterized under identical ionic conditions with low internal Mg(2+). Removal of internal Mg(2+) induced MIC current despite widely varying Ca(2+) and EGTA levels, suggesting that Ca(2+)-store depletion is not involved in activation of MIC channels. Increasing internal Mg(2+) from submicromolar to millimolar levels decreased MIC currents without affecting rectification but did not alter CRAC current rectification or amplitudes. External Mg(2+) and Cs(+) carried current through MIC but not CRAC channels. SKF-96365 blocked CRAC current reversibly but inhibited MIC current irreversibly. At micromolar concentrations, both spermine and extracellular Mg(2+) blocked monovalent MIC current reversibly but not monovalent CRAC current. The biophysical characteristics of MIC current match well with cloned and expressed TRPM7 channels. Previous results are reevaluated in terms of separate CRAC and MIC channels.     

THE LACK OF SPECIFICITY TOWARDS SALTS IN THE ACTIVATION OF CHOLINE ACETYLTRANSFERASE FROM HUMAN PLACENTA

Abstract— The effects of monovalent and divalent anions on the choline acetyltransferase reaction have been determined at high (5.0 mM) and low (0.58 mM) choline. At 0.58 mM-choline, both monovalent and divalent anions activate the enzyme ±9 fold; however, at 5.0mM-choline, monovalent anions activate the enzyme ±25 fold, while divalent anions activate ±9 fold. Both monovalent and divalent anions show uncompetitive activation with respect to choline. When either dimethylaminoethanol, N -(2-hydroxyethyl)- N -methyl piperidinium iodide, or N -(2-hydroxyethyl)- N -propyl pyrrolidinium iodide was substituted for choline, activation by monovalent or divalent anions was only 2.5-4 fold. With AcCoA as substrate the ChA reaction can be increased ±20 fold by increased salts; however, with acetyl dephosphoCoA as substrate, the reaction is insensitive to the salt concentration. Similar salt effects on the ChA reaction, as measured in the direction of acetylcholine synthesis, have been demonstrated in the reverse reaction. In addition, inhibition of the forward reaction by acetylcholine has been measured as a function of sodium chloride concentration. Although the K₁ for acetylcholine increases with increasing salt, this change in K ₁, parallels the increase in the K _m for choline. These results support the hypothesis that both monovalent and divalent anions activate choline acetyltransferase by the same singular mechanism; which is to increase the rate of dissociation of coenzyme A from the enzyme.