A new approach to enhance PCR specificity

A new approach to enhanced specificity and product yield of polymerase chain reaction is proposed. It is based on control of DNA polymerase activity during PCR by changing the magnesium ion concentration, which depends on the temperature of the reaction mixture. A slightly soluble magnesium salt, magnesium oxalate, whose solubility depends on temperature, was used as a source of magnesium ions. During PCR, magnesium oxalate was maintained at saturating concentration by the presence of an insoluble excess of this salt, and the concentration of magnesium ions depended on the salt solubility: binding of magnesium ions at lower temperatures and their release at higher temperatures was shown to affect the DNA polymerase activity and to favor the specific PCR amplification of the target DNA fragment.     

In vitro repair of gamma-irradiated transforming Bacillus subtilis DNA by extracts of blue-green algae

A cell-free extract from blue-green alga Anacystis nidulans contains enzymes which repair in vitro the transforming activity of gamma-irradiated Bacillus subtilis DNA. The level of restoration of the transforming activity depends on the protein concentration in the reaction mixture, the duration of incubation and on the dose of irradiation. The repair of gamma-induced lesions is most efficient in the presence of magnesium ions, NAD and ATP. The present data indicate that the repair of transforming DNA is performed with the participation of DNA polymerase and polynucleotide ligase which function in the cell-free extract of algae.     

Dissolution and growth of calcium oxalate monohydrate I. Effect of magnesium and pH

The rate of dissolution of calcium oxalate monohydrate and of a calcium oxalate renal stone was measured in 0.9% NaCl solution at different levels of magnesium concentration and pH. The growth of calcium oxalate obtained by chemical reaction between Ca²⁺ and oxalate ions at a concentration similar to that existing in normal urine was also investigated as a function of pH and magnesium concentration. It was found that both magnesium and pH exert a fine kinetic control on the precipitation and growth of calcium oxalate monohydrate. Magnesium had no effect on the dissolution. The possible role of magnesium and pH in calcium oxalate urolithiasis has been discussed in the light of previous reports and of the data presented in this study.     

快速可靠的双链PCR产物直接测序法

利用T7DNA聚合酶在低温下仍具较高活性的特点,在热变性后低温下进行测序反应,使用该方法对多种PCR产物进行序列分析均取得较好的结果．     

Microcalorimetric study of heat denaturation of DNA from calf thymus DNA in the absence of magnesium ions

Thermal denaturation was studied for a wide range of magnesium ions concentrations and salt concentration 0.15 M NaCl. It was shown that thermal stability of DNA increases at low Mg/2P ratios and decreases at high concentrations of magnesium ions. Up to Mg/2P = 10 DNA denaturation is an equilibrium process. With an increase in magnesium ions concentrations the enthalpy of DNA denaturation reaches the maximum at Mg/2P = 10 (50 kJ/mole base pairs). DNA aggregation and appearance of a new heat absorption peak is observed in the high temperature region at Mg/2P = 10. At this region of magnesium ions concentrations DNA denaturation process is non-equilibrium.     

Polymerase chain reaction (PCR) detection of Listeria monocytogenes in diluted milk and reversal of PCR inhibition caused by calcium ions

J. BICKLEY, J. K. SHORT, D. G. MCDOWELL AND H. C. PARKES. 1996. DNA from Listeria monocytogenes was used as the model system from this investigation, with PCR primers based on the listeriolysin O gene. Under standard polymerase chain reaction (PCR) conditions and with no prior treatment, amplification failed in the presence of more than 5% milk. Since inhibition of the PCR occurred at the same milk concentrations with full fat, half fat and fat-free milk, inhibition was not attributed to the fat content of the milk. Calcium ions were, however, identified as a major source of PCR inhibition. The results demonstrated that the inhibitory effects of calcium ions and milk could be partially reversed by increasing the magnesium concentration in the reaction to well above the standard levels normally required for PCR. This work has important implications for the use of the PCR in the direct detection of food pathogens.     

Purification and characterization of a common soil component which inhibits the polymerase chain reaction

DNA prepared from soil usually contains a brown-tinted inhibitor of the polymerase chain reaction (PCR) which limits the sensitivity of this technique for specific detection of microorganisms. To localize the inhibitor, soil fractions were tested for their inhibitory effect on the PCR reaction. A highly inhibitory activity, sufficient to account for the inhibition typically exhibited by soil DNA, was found to be tightly associated with the soil microorganism fraction. After cell breakage, the inhibitory material became soluble, and was not separable from DNA by standard purification procedures. A method was derived by which most of the inhibitory material could be selectively solubilized from the microorganism fraction without cell breakage, using successive washes with buffers differing in EDTA concentration. This technique was used to isolate a substance with characteristics suggesting that it is the major PCR inhibitor contaminating DNA purified from soil. It was found to be an organic, water-soluble compound of high molecular weight, and was present in a variety of soil types from different locations. It was found to be distinctly different in its solubility properties from humic and fulvic acids, and also in its FT-IR and NMR spectra. It forms a complex with protein and may inhibit the PCR reaction by an interaction with Taq DNA polymerase.     

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.     

In vitro repair of UV-or X-irradiated bacteriophage T4 DNA by extract from blue-green alga Anacystis nidulans

The cell-free extract from blue-green alga Anacystis nidulans contains enzymatic activities which repair in vitro transforming DNA of bacteriophage T4 damaged by UV light or X-rays. The repair effect of the extract was observed with double-stranded irradiated DNA but not with denatured irradiated DNA. The level of restoration of the transforming activity depends on the protein concentration in the reaction mixture and on the dose of irradiation. A fraction of DNA lesions induced by X-rays is repaired by a NAD-dependent polynucleotide ligase present in the extract. The repair of UV-induced lesions is the most efficient in the presence of magnesium ions, NAD, ATP and the four deoxynucleoside triphosphates. The results indicate that the repair of UV-irradiated DNA is performed with the participation of DNA polymerase and polynucleotide ligase which function in the cell-free extract of the algae on the background of a low deoxyribonuclease activity.Abbreviations UV ultraviolet - TA transforming activity - PN-ligase polynucleotide ligase - NAD nicotinamide adenine dinucleotide - dNTP deoxynucleoside triphosphates - dATP, dGTP, dTTP triphosphates of deoxyadenosine, deoxyguanosine, deoxythymidine and deoxycytidine, respectively     

DNA polymerases of ascites hepatoma cells. I. Purification and properties of a DNA polymerase from soluble fraction

DNA polymerase from soluble fraction of ascites hepatoma cells has been purified about 490-fold. The polymerase requires template DNA, all four deoxyribonucleoside triphosphates, and magnesium ions for the reaction. Optimal activity was found at pH 7.0 – 7.5, with 3 – 8 mM magnesium chloride, and 20 – 40 mM potassium phosphate. The purified enzyme utilizes preferentially DNA treated with pancreatic DNase as template.     

Following ionic activity by electrochemistry during the polymerase chain reaction

The most commonly used technique for gene detection is the polymerase chain reaction (PCR). PCR is associated with alterations in ionic activity because inorganic pyrophosphate (PPi) and inorganic phosphate (Pi) ions are produced during nucleotide polymerization. To maintain electro-neutrality, magnesium, potassium, and ammonium ions are bound to DNA. Deoxynucleotides are also bound to DNA during PCR. Some authors have described DNA itself as an electrically conducting polymer formed by base stapling with the formation of extensive Pi systems. In the current study, alterations in electrical conductivity determined experimentally during PCR are reported, and a model explaining the observed changes is described. During recent years, several different techniques for quantifying PCR products have been developed. The most frequently used technique is comparison of the densitometric intensities of ethidium bromide-stained PCR products separated by electrophoresis on gels. Here an alternative technique for quantifying PCR products by measuring alterations in electrical conductivity during PCR is presented.     

Temperature effect on polymerase fidelity

The discovery of extremophiles helped enable the development of groundbreaking technology such as PCR. Temperature variation is often an essential step of these technology platforms, but the effect of temperature on the error rate of polymerases from different origins is underexplored. Here, we applied high-throughput sequencing to profile the error rates of DNA polymerases from psychrophilic, mesophilic, and thermophilic origins with single-molecule resolution. We found that the reaction temperature substantially increases substitution and deletion error rates of psychrophilic and mesophilic DNA polymerases. Our motif analysis shows that the substitution error profiles cluster according to phylogenetic similarity of polymerases, not the reaction temperature, thus suggesting that the reaction temperature increases the global error rate of polymerases independent of the sequence context. Intriguingly, we also found that the DNA polymerase I of psychrophilic bacteria exhibits higher polymerization activity than its mesophilic ortholog across all temperature ranges, including down to −19 ^°C, which is well below the freezing temperature of water. Our results provide a useful reference for how the reaction temperature, a crucial parameter of biochemistry, can affect DNA polymerase fidelity in organisms adapted to a wide range of thermal environments.     

Influence of magnesium ions on helix-coil transition of DNA determined by modified differential scanning calorimeter

The thermal effect of magnesium ions on the helix–coil transition of DNA was studied calorimetrically by a modified differential scanning calorimeter (DSC). It was found that the transition temperature of DNA depends on both the DNA and magnesium ion concentrations. The dependence of the helix–coil transition of DNA on the mole ratio of magnesium ions to DNA(P) can be classified into two groups. When this mole ratio is less than 1, magnesium ions tend to stabilize the double-helix DNA, so that the transition temperature increases linearly and the heat of transition increases significantly with increasing mole ratio. When the mole ratio is more than 1, magnesium ions tend to destabilize the double-helix DNA, so that DNA precipitates when the temperature is raised above the transition temperature. In this case, both the transition temperature and the heat of transition decrease with increasing mole ratio.     

唐古特大黄ISSR-PCR反应条件的优化

利用单因素试验对影响唐古特大黄ISSR-PCR扩增的重要参数进行优化,以期建立其最佳反应条件。结果如下:20μL反应体系包括1.5×PCR buffer(15mmol/LTris-HCl,75mmol/LKCl),1.00mmol/LMgCl2,0.6UTaq DNA聚合酶,0.125mmol/LdNTP,0.5μmol/L引物和30ng模板DNA;引物UBC888适宜的退火温度为57.4℃。ISSR反应条件的建立为利用分子标记技术研究唐古特大黄居群遗传多样性奠定了良好基础。     

Protection of DNA by salts against thermodegradation at temperatures typical for hyperthermophiles

The effect of physiological concentrations of KCl and MgCl₂ on the chemical stability of double-stranded and single-stranded DNA has been studied at temperatures typical for hyperthermophiles. These two salts protect both double and single-stranded DNA against heat-induced cleavage by inhibiting depurination. High KCl concentrations also protect DNA cleavage at apurinic sites, while high MgCl₂ concentrations stimulate this cleavage. It has been previously proposed that salt protects double-stranded DNA against depurination by stabilizing the double helix. However, the inhibition of the depurination of single-stranded DNA by KCl and MgCl₂ indicates that this effect is more probably due to a direct interaction of salts with purine nucleotides. These results suggest that the number and nature of heat-induced DNA lesions which have to be repaired might be quite different from one hyperthermophile to another, depending on their intracellular salt concentration. High salt concentrations might be also useful to protect DNA in long polymerase chain reaction (PCR) experiments and for long-term preservation. Received: October 12, 1997 / Accepted: January 29, 1998     

Guidelines for the Tetra-Primer ARMS–PCR Technique Development

The tetra-primer amplification refractory mutation system–polymerase chain (ARMS–PCR) reaction is a simple and economical method to genotype single-nucleotide polymorphisms (SNPs). It uses four primers in a single PCR and is followed just by gel electrophoresis. However, the optimization step can be very hardworking and time-consuming. Hence, we propose to demonstrate and discuss critical steps for its development, in a way to provide useful information. Two SNPs that provided different amplification conditions were selected. DNA extraction methods, annealing temperatures, PCR cycles protocols, reagents, and primers concentration were also analyzed. The use of tetra-primer ARMS–PCR could be impaired for SNPs in DNA regions rich in cytosine and guanine and for samples with DNA not purified. The melting temperature was considered the factor of greater interference. However, small changes in the reagents concentration significantly affect the PCR, especially MgCl₂. Balancing the inner primers band is also a key step. So, in order to balance the inner primers band, intensity is important to observe which one has the weakest band and promote its band by increasing its concentration. The use of tetra-primer ARMS–PCR attends the expectations of modern genomic research and allows the study of SNPs in a fast, reliable, and low-cost way.     

A DNA topoisomerase activity copurifies with the DNA polymerase induced by herpes simplex virus

A DNA-relaxing enzyme was found to copurify along with herpes simplex virus type I (HSV-1)-induced DNA polymerase throughout a multistep purification scheme. Both the enzymes had similar sedimentation velocity, required high ionic strength for optimal enzymatic activities and showed time dependence of reaction. The DNA-relaxing enzyme however, differed from the HSV-1 DNA polymerase in its requirement for higher Mg²⁺ concentration, rATP and much broader pH dependence. Furthermore, phosphonoacetic acid, a potent inhibitor of HSV-1 DNA polymerase did not influence the DNA-relaxing activity even at a much higher concentration. On the other hand, the DNA-relaxing enzyme associated with the DNA polymerase may be specified by HSV-1 since IgG fraction of rabbit antisera against the virus-infected cells but not against the mock-infected cells strongly inhibited both the enzymatic activities. Thus, HSV-1-induced DNA polymerase which is known to be associated with a 3′ to 5′ exonuclease may also be associated with yet another enzymatic activity involved in DNA metabolism.     

Fluorescence-based temperature control for polymerase chain reaction

The ability to accurately monitor solution temperature is important for the polymerase chain reaction (PCR). Robust amplification during PCR is contingent on the solution reaching denaturation and annealing temperatures. By correlating temperature to the fluorescence of a passive dye, noninvasive monitoring of solution temperatures is possible. The temperature sensitivity of 22 fluorescent dyes was assessed. Emission spectra were monitored and the change in fluorescence between 45 and 95 °C was quantified. Seven dyes decreased in intensity as the temperature increased, and 15 were variable depending on the excitation wavelength. Sulforhodamine B (monosodium salt) exhibited a fold change in fluorescence of 2.85. Faster PCR minimizes cycling times and improves turnaround time, throughput, and specificity. If temperature measurements are accurate, no holding period is required even at rapid speeds. A custom instrument using fluorescence-based temperature monitoring with dynamic feedback control for temperature cycling amplified a fragment surrounding rs917118 from genomic DNA in 3 min and 45 s using 35 cycles, allowing subsequent genotyping by high-resolution melting analysis. Gold-standard thermocouple readings and fluorescence-based temperature differences were 0.29 ± 0.17 and 0.96 ± 0.26 °C at annealing and denaturation, respectively. This new method for temperature cycling may allow faster speeds for PCR than currently considered possible.     

A Mutation in the gene-encoding bacteriophage T7 DNA polymerase that renders the phage temperature-sensitive

Gene 5 of bacteriophage T7 encodes a DNA polymerase essential for phage replication. A single point mutation in gene 5 confers temperature sensitivity for phage growth. The mutation results in an alanine to valine substitution at residue 73 in the exonuclease domain. Upon infection of Escherichia coli by the temperature-sensitive phage at 42 degrees C, there is no detectable T7 DNA synthesis in vivo. DNA polymerase activity in these phage-infected cell extracts is undetectable at assay temperatures of 30 degrees C or 42 degrees C. Upon infection at 30 degrees C, both DNA synthesis in vivo and DNA polymerase activity in cell extracts assayed at 30 degrees C or 42 degrees C approach levels observed using wild-type T7 phage. The amount of soluble gene 5 protein produced at 42 degrees C is comparable to that produced at 30 degrees C, indicating that the temperature-sensitive phenotype is not due to reduced expression, stability, or solubility. Thus the polymerase induced at elevated temperatures by the temperature-sensitive phage is functionally inactive. Consistent with this observation, biochemical properties and heat inactivation profiles of the genetically altered enzyme over-produced at 30 degrees C closely resemble that of wild-type T7 DNA polymerase. It is likely that the polymerase produced at elevated temperatures is a misfolded intermediate in its folding pathway.