Effects of fluorescent dyes, quenchers, and dangling ends on DNA duplex stability

Moreira, Bernardo G.; You, Yong; Behlke, Mark A.; Owczarzy, Richard

doi:10.1016/j.bbrc.2004.12.035

Cited by 138 publications

(134 citation statements)

References 34 publications

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“…It was also observed that beyond 3.5 mM concentration of MgCl 2 no amplification was observed (Table 1). Our findings were in accordance with the results of Moreira et al, [21] who reported that too much MgCl 2 is equally deleterious in most cases. In the case of excess Mg 2+ , the usual effect is a substantial increase in Table 1 Effect of different concentration of MgCl 2 on the specificity of the goat specific primer G. secondary products' formation by non-specific priming.…”

Section: Standardisation Of Pcr Conditions To Increase the Specificitsupporting

confidence: 94%

A species specific simplex polymerase chain reaction-based approach for detection of goat tallow in heat clarified milk fat (ghee)

Hazra

Sharma

et al. 2017

International Journal of Food Properties

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The detection of milk fat adulteration with foreign fats/oils continues to be a challenge for the dairy industry as well as food testing laboratories. Recently, various chromatographic methods have been developed to detect a low level of vegetable oil in ghee but detection of animal body fat in ghee is still a challenge. In India, goat body fat (goat tallow) adulteration in ghee is a very serious problem. In the present investigation, a rapid species specific polymerase chain reaction (PCR)-based methodology has been standardised to detect goat tallow in heat clarified milk fat (ghee). This standardised PCR-based protocol did not show any false positive results in the genuine ghee (heat clarified butter fat) samples. Adulteration of ghee (both cow and buffalo) with goat tallow at the 10% level could be detected using the standardised PCR-based protocol. The protocol standardised is rapid and convenient to use. ARTICLE HISTORY

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Section: Standardisation Of Pcr Conditions To Increase the Specificitsupporting

confidence: 94%

A species specific simplex polymerase chain reaction-based approach for detection of goat tallow in heat clarified milk fat (ghee)

Hazra

Sharma

et al. 2017

International Journal of Food Properties

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“…A complexometric assay (34) was employed to verify Mg 2+ concentrations in each lot of the buffer. EDTA titrations showed that magnesium concentrations deviated less than 2% from expected values.…”

Section: Methodsmentioning

confidence: 99%

Predicting Stability of DNA Duplexes in Solutions Containing Magnesium and Monovalent Cations

et al. 2008

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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.Interactions of magnesium, sodium and potassium ions with DNA 1 and RNA molecules are important for essential functions of living cells and for molecular biology applications. Both divalent and monovalent cations bind to nucleic acid molecules and affect their physical properties (1). Magnesium cations stabilize nucleic acid duplexes and facilitate their folding into secondary and tertiary structures (2), which are biologically active. Mg 2+ ions are also necessary cofactors of many enzymatic reactions involving nucleic acids (3). The total magnesium concentrations in various cells range from 5 to 30 mM; however, free Mg 2+ concentrations are tightly controlled between 0.4 and 1.2 mM (4). Binding of monovalent cations also increases duplex stability. The major intracellular monovalent cation is K + , while the major monovalent cation in extracellular fluid is Na + .Several theoretical models have been proposed to describe the complex phenomena of associations between cations and nucleic acids. The most widely used approaches are the mean-field approximation of the Poisson-Boltzmann equation (5-8), the counterion condensation model (9, 10), and Monte Carlo simulations (11,12). There have been few experimental studies addressing effects of Mg 2+ ion on the stability of short DNA duplexes (13-16), and comprehensive experimental melting data are not available. The majority of melting studies in magnesium buffers investigated genomic DNAs (e.g., calf thymus) (5,(17)(18)(19), long polymeric repeats (20-22), or biologically active RNA molecules (2, 23). Pione...

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“…Substitution into the FreeSRF relationship, ρ pred = χ model β exp C, yields Eq. 7: ρ c = 78.1 RIU mM × 1,055,663 milliradians RIU × CðMÞ, [7] which allows the ρ c at each ligand concentration to be calculated.…”

Section: Conformation and Hydration Changes Are The Origin Of Free-somentioning

confidence: 99%

“…Label-free chemical and biochemical investigations (5, 6) transduce the desired signal without an exogenous label (fluorescent, radioactive, or otherwise) representing an essential step toward this goal. Many label-free methods require one of the interacting species to be either tethered or immobilized to the sensor surface, introducing a potential perturbation to the natural state of the species (7,8). However, back-scattering interferometry (BSI) is a free-solution label-free technique with the added benefit of sensitivity that rivals fluorescence (9).…”

mentioning

confidence: 99%

Origin and prediction of free-solution interaction studies performed label-free

Bornhop

Kammer

Kussrow

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Interaction/reaction assays have led to significant scientific discoveries in the biochemical, medical, and chemical disciplines. Several fundamental driving forces form the basis of intermolecular and intramolecular interactions in chemical and biochemical systems (London dispersion, hydrogen bonding, hydrophobic, and electrostatic), and in the past three decades the sophistication and power of techniques to interrogate these processes has developed at an unprecedented rate. In particular, label-free methods have flourished, such as NMR, mass spectrometry (MS), surface plasmon resonance (SPR), biolayer interferometry (BLI), and backscattering interferometry (BSI), which can facilitate assays without altering the participating components. The shortcoming of most refractive index (RI)-based label-free methods such as BLI and SPR is the requirement to tether one of the interaction entities to a sensor surface. This is not the case for BSI. Here, our hypothesis is that the signal origin for freesolution, label-free determinations can be attributed to conformation and hydration-induced changes in the solution RI. We propose a model for the free-solution response function (FreeSRF) and show that, when quality bound and unbound structural data are available, FreeSRF correlates well with the experiment (R 2 > 0.99, Spearman rank correlation coefficients >0.9) and the model is predictive within ∼15% of the experimental binding signal. It is also demonstrated that a simple mass-weighted dη/dC response function is the incorrect equation to determine that the change in RI is produced by binding or folding event in free solution.backscattering interferometry | assay methodology | molecular interactions | conformation change | hydration change C ontemporary assays enabling single-molecule detection (1,2) have accelerated the sequencing of the human genome (3) and facilitated imaging with extraordinary resolution without labels (4). To most closely approximate the natural state, an interaction assay methodology would interrogate the processes (reaction, molecular interaction, protein folding event, etc.) without perturbation. Label-free chemical and biochemical investigations (5, 6) transduce the desired signal without an exogenous label (fluorescent, radioactive, or otherwise) representing an essential step toward this goal. Many label-free methods require one of the interacting species to be either tethered or immobilized to the sensor surface, introducing a potential perturbation to the natural state of the species (7,8). However, back-scattering interferometry (BSI) is a free-solution label-free technique with the added benefit of sensitivity that rivals fluorescence (9). There are other techniques performed in free solution, such as MS (10, 11) and NMR (12,13) and the widely used isothermal titration calorimetry (ITC) (14, 15). As with NMR, ITC has many advantages, but exhibits modest sensitivity and often requires large sample quantities. Another increasingly popular free-solution approach is microscale thermophoresis (...

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Effects of fluorescent dyes, quenchers, and dangling ends on DNA duplex stability

Cited by 138 publications

References 34 publications

A species specific simplex polymerase chain reaction-based approach for detection of goat tallow in heat clarified milk fat (ghee)

A species specific simplex polymerase chain reaction-based approach for detection of goat tallow in heat clarified milk fat (ghee)

Predicting Stability of DNA Duplexes in Solutions Containing Magnesium and Monovalent Cations

Origin and prediction of free-solution interaction studies performed label-free

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