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  • 1
    Electronic Resource
    Electronic Resource
    s.l. : American Chemical Society
    Analytical chemistry 66 (1994), S. 3416-3422 
    ISSN: 1520-6882
    Source: ACS Legacy Archives
    Topics: Chemistry and Pharmacology
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 0006-3525
    Keywords: Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: Seventeen DNA dumbbells were constructed that have duplex sequences ranging in length from 14 to 18 base pairs linked on the ends by T4 single-strand loops. Fifteen of the molecules have the core duplexes with the sequences 5′G-T-A-T-C-C-(W-X-Y-Z)-G-G-A-T-A-C3′, where (W-X-Y-Z) represents a unique combination of A · T, T · A, G · C, and C · G base pairs. The remaining two molecules have the central sequences (W-X-Y-Z) = A-C and A-C-A-C-A-C. These duplex sequences were designed such that the central sequences include different combinations of the 10 possible nearest-neighbor (n-n) stacks in DNA. In this sense the set of molecules is complete and serves as a model system for evaluating sequence-dependent local stability of DNA. Optical melting curves of the samples were collected in 25, 55, 85, and 115 mM [Na +], and showed, regardless of solvent ionic strength, that the transition temperatures of the dumbbells vary by as much as 14° for different molecules of the set.Results of melting experiments analyzed in terms of a n-n sequence-dependent model allowed evaluation of nine independent linear combinations of the n-n stacking interactions in DNA as a function of solvent ionic strength. Although there are in principle 10 possible different n-n interactions in DNA, these 10 are not linearly independent and therefore can not be uniquely determined. For molecules with ends, there are 9 linearly independent combinations, as opposed to circular or semiinfinite repeating copolymers where only 8 linear combinations of the 10 possible n-n interactions are linearly independent. The n-n interactions are presented as combinations of the deviations from average stacking for the 5′-3′ base-pair doublets, δGi, and reveal several interesting features: (1) Titratable changes in the values of δGi, with changing salt environment are observed. In all salts the most stable unique combination is δG4 = (δGGpC + δGCpG,)/2, and the least stable is the GpG/CpC stack, δG2 = δGGpG/CpC. (2) The χ2 values of the fits of the evaluated δGi's to experimental data increased with decreasing [Na +], suggesting that significant interactions beyond nearest neighbors become more pronounced, particularly at 25 mM Na +. (3) In 85 and 115 mM Na +, where the n-n approximation seems to be most valid, the absolute value of δGi for any n-n stack or average of two n-n stacks is not more than ∼ 220 cal/mole, indicating that deviations from average stacking due to n-n interactions represent about 15% of the total stability of a base pair. The overall thermodynamic stability of DNA is predominantly determined by the sequence content (%G · C). Even though the contribution of n-n interactions to overall stability are intrinsically small, reliable predictions of DNA transition temperatures de novo from sequence can be significantly compromised by cumulative errors in the δGi's. (4) Comparisons of our set of n-n linear combinations evaluated in 115 mM Na+ with various published sets evaluated from melting experiments of long restriction fragments, synthetic polymers, and short oligomers, and those obtained from a reanalysis of published melting data of synthetic polymers, are presented. The analysis reveals a major consensus agreement between n-n free energies evaluated from melting data of restriction fragments and long synthetic repeating copolymers. In contrast, only a minor consensus agreement is obtained between our n-n set and these values or those obtained from melting analysis of a combination of short oligomers and long polymers or those theoretically calculated. Results of these comparisons suggest the values of n-n interactions evaluated from DNA melting curves depend on the length of the melted duplex regions of the DNA molecules that comprise the sample set.
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  • 3
    ISSN: 0006-3525
    Keywords: Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: The preparation and melting of a 16 base-pair duplex DNA linked on both ends by C12H24 (dodecyl) chains is described. Absorbance vs temperature curves (optical melting curves) were measured for the dodecyl-linked molecule and the same duplex molecule linked on the ends instead by T4 loops. Optical melting curves of both molecules were measured in 25, 55, and 85 mM Na+ and revealed, regardless of [Na +], the duplex linked by dodecyl loops is more stable by at least 6°C than the same duplex linked by T4 loops. Experimental curves in each salt environment were analyzed in terms of the two-state and multistate theoretical models. In the two-state, or van't Hoff analysis, the melting transition is assumed to occur in an all-or-none manner. Thus, the only possible states accessible to the molecule throughout the melting transition are the completely intact duplex and the completely melted duplex or minicircle. In the multistate analysis no assumptions regarding the melting transition are required and the statistical occurrence of every possible partially melted state of the duplex is explicitly considered. Results of the analysis revealed the melting transitions of both the dodecyl-linked molecule and the dumbbell with T4 end loops are essentially two state in 25 and 55 mM Na+. In contrast, significant deviations from two-state behavior were observed in 85 m MNa+. From our previously published melting data of DNA dumbbells with Tn end loops where n = 2, 3, 4, 6, 8, 10, 14 [T. M. Paner, M. Amaratunga, and A. S. Benight, (1992) Biopolymers, Vol. 32, pp. 881-892] and the dumbbell with T4 end loops of this study, a plot of d(Tm)/d ln [Na+] was constructed. Extrapolation of this data to n = 1 intersects with the value of d (Tm)/d ln [Na+] obtained for the alkyl-linked dumbbell, suggesting the salt-dependent stability of the alkyl-linked molecule behaves as though the duplex of this molecule were linked by end loops comprised of a single T residue. © 1993 John Wiley & Sons, Inc.
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  • 4
    ISSN: 0006-3525
    Keywords: Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: Optical melting transitions of the short DNA hairpins formed from the self-complementary DNA oligomers d[GGATACX4GTATCC] where X = A, T, G, or C measured in 100 mM NaCl are presented. A significant dependence of the melting transitions on loop sequence is observed and transition temperatures, tm, of the hairpins vary from 58.3°C for the T4 loop hairpin to 55.3°C for the A4 loop. A nearest-neighbor sequence-dependent theoretical algorithm for calculating melting curves of DNA hairpins is presented and employed to analyze the experimental melting transitions. Experimental melting curves were fit by adjustment of a single theoretical parameter, Fend(n), the weighting function for a hairpin loop comprised of n single-strand bases. Empirically determined values of Fend(n) provide an evaluation of the free-energy of hairpin loop formation and stability. Effects of heterogeneous nearest-neighbor sequence interactions in the duplex stem on hairpin loop for mation were investigated by evaluating Fend(n) in individual fitting procedures using two of the published sets of nearest-neighbor stacking interactions in DNA evaluated in 100 mM NaCl and given by Wartell and Benight, 1985. In all cases, evaluated values of Fend(n) were obtained that provided exact theoretical predictions of the experimental transitions.Results of the evaluations indicate: (1) Evaluated free-energies of hairpin loop formation are only slightly dependent on loop sequences examined. At the transition temperature, Tm, the free-energy of forming a loop of four bases is approximately equal for T4, G4, or C4 loops and varies from 3.9 to 4.8 kcal/mole depending on the set of nearest-neighbor interactions employed in the evaluations. This result suggests, in light of the observed differences in stability between the T4, G4, and C4 loop hairpins, that sequence-dependent interactions between base residues of the loop are most likely not the source of the enhanced stability of a T4 loop. In contrast, the evaluated free-energy of forming an A4 loop is approximately 400 cal/mole higher for each nearest-neighbor set indicating unfavorable interactions between A bases in a loop-affect loop formation and overall hairpin stability, (2) The absolute value for the free-energy of loop formation at the Tm of each hairpin varies by about 1 kcal/mole depending on the set of nearest-neighbor interactions employed and the relative hierarchy of stability for each loop is conserved for different nearest neighbor sets, (3) The melting process of each hairpin deviates from strict two-state behavior in the order according to loop sequence of T 〉 A 〉 G 〉 C, (4) Results of our analysis are compared with the early work of Scheffler et al., 1970 on the hairpins formed from the copolymer sequences d(T - A)q where q = 9-21. Comparisons with the more recent works a DNA dumbbell (Benight et al., 1988) and the very similar DNA hairpins studied by Senior et al., 1988 are presented.
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  • 5
    ISSN: 0006-3525
    Keywords: Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: Expressions for the partition function Q(T) of DNA hairpins are presented. Calculations of Q(T), in conjunction with our previously reported numerically exact algorithm [T. M. Paner, M. Amaratunga, M. J. Doktycz, and A. S. Benight (1990) Biopolymers, 29, 1715-1734], yield a numerical method to evaluate the temperature dependence of the transition enthalpy, entropy, and free energy of a DNA hairpin directly from its optical melting curve. No prior assumptions that the short hairpins melt in a two-state manner are required. This method is then applied in a systematic manner to investigate the stability of the six base-pair duplex stem 5′-GGATAC-3′ having four-base dangling single-strand ends with the sequences (XY)2, where X, Y = A, T, G, C, on the 5′ end and a T4 loop on the 3′ end.Results show that all dangling ends of the sample set stabilize the hairpin against melting. Increases in transition temperatures as great as 4.0°C above the blunt-ended control hairpin were observed. The hierarchy of the hairpin transition temperatures is dictated by the identity of the first base of the dangling end adjoining the duplex in the order: purine 〉 T 〉 C. Calculated melting curves of every hairpin were fit to experimental curves by adjustment of a single parameter in the numerically exact theoretical algorithm. Exact fits were obtained in all cases. Experimental melting curves were also calculated assuming a two-state melting process. Equally accurate fits of all dangling-ended hairpin melting curves were obtained with the two-state model calculation. This was not the case for the melting curve of the blunt-ended hairpin, indicating the presence of a four-base dangling-end drives hairpin melting to a two-state process. Q(T) was calculated as a function of temperature for each hairpin using the theoretical parameters that provided calculated curves in exact agreement with the experimentally obtained optical melting curves. From Q(T), the temperature dependence of the transition enthply ΔH, enytropy ΔS, and free energy ΔG were calculated for every hairpin providing a quantitative assessment of the effects of dangling ends on hairpin thermodynamics. Comparisons of our results are made with those of the Breslauer group [M. Senior, R. A. Jones, and K. J. Breslauer (1988) Biochemistry 27, 3879-3885] on the T25′ dangling-ended d (GC)3 duplexes.To estimate the average contribution to stability of each single-stand nearest neighbor stack to the duplex stem, the relative values ΔΔH and ΔΔS of the transition enthalpy and entropy for each dangling-ended hairpin compared to the blunt-ended control hairpin were cast in a system of 16 equation in the 16 unkowns, The nonsingular system of equations was solved for the unknowns by matrix diagonalization, which yielded the relative average contribution of each of the 16 possible nearest neighbor dinucleotide 5′3′ stacks in single strand DNA to the stability of a 5′-GGATAC-3′ duplex stem.
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  • 6
    ISSN: 0006-3525
    Keywords: Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: CD spectra and melting curves were collected for a 28 base-pair DNA fragment in the form of a DNA dumbbell (linked on both ends by T4 single-strand loops) and the same DNA sequence in the linear form (without end loops). The central 16 base pairs (bp) of the 28-bp duplex region is the poly(pu) sequence: 5′-AGGAAGGAGGAAAGAG-3′. Mixtures of the dumbbell and linear DNAs with the 16-base single-strand sequence 5′-TCCTTCCTCCTTTCTC-3′ were also prepared and studied. At 22°C, CD measurements of the mixtures in 950 mM NaCl, 10 mM sodium acetate, 1 mM EDTA, pH 5.5, at a duplex concentration of 1.8 μM, provided evidence for triplex formation. Spectroscopic features of the triplexes formed with either a dumbbell or linear substrate were quite similar. Melting curves of the duplex molecules alone and in mixtures with the third strand were collected as a function of duplex concentration from 0.16 to 2.15 μM. Melting curves of the dumbbell alone and mixtures with the third strand were entirely independent of DNA concentration. In contrast, melting curves of the linear duplex alone or mixed with the third strand were concentration dependent. At identical duplex concentrations, the dumbbell alone melts ∼20°C higher than the linear duplex. The curve of the linear duplex displayed a significant pretransition probably due to end fraying.On melting curves of mixtures of the dumbbell or linear duplex with the third strand, a low temperature transition with much lower relative hyperchromicity change (∼ 5%) was observed. This transition was attributed to the melting of a new molecular species, e.g., the triplex formed between the duplex and single-strand DNA molecules. In the case of the dumbbell/single-strand mixture, these melting transitions of the triplex and the dumbbell were entirely resolvable. In contrast, the melting transitions of the linear duplex and the triplex overlapped, thereby preventing their clear distinction. To analyze the data, a three-state equilibrium model is presented. The analysis utilizes differences in relative absorbance vs temperature curves of dumbbells (or linear molecules) alone and in mixtures with the third strand. From the model analysis a straightforward derivation of fT(T), the fraction of triplex as a function of temperature, was obtained. Analysis of fT vs temperature curves, in effect melting curves of the triplexes, provided evaluation of thermodynamic parameters of the melting transition. For the triplex formed with the dumbbell substrate, the total transition enthalpy is ΔHT = 118.4 ± 12.8 kcal/mol (7.4 ± 0.8 kcal/mol per triplet unit) and the total transition entropy is ΔST = 344 ± 36.8 cal/K · mol (eu) (21.5 ± 2.3 eu per triple unit). The transition curves of the triplex formed with the linear duplex substrate displayed two distinct regions. A broad pretransition region from fT = 0 to 0.55 and a higher, sharper transition above fT = 0.55. The transition parameters derived from the lower temperature region of the curve are ΔH′T = 44.8 ± 9.6 kcal/mol and ΔS′T = 112 ± 33.6 eu (or ΔH′ = 2.8 ± 0.6 kcal/mol and ΔS′ = 7.0 ± 2.1 eu per triplet). These values are probably too small to correspond to actual melting of the triplex but instead likely reveal effects of end fraying of the duplex substrate on triplex stability. Transition parameters of the upper transition are ΔH′T = 128.0 ± 2.3 kcal/mol and ΔS′T = 379.2 ± 6.4 eu (ΔH′ = 8.0 ± 0.2 kcal/mol and ΔS′ = 23.7 ± 0.4 eu per triplet) in good agreement (within experimental error) with the transition parameters of the triplex formed with the dumbbell substrate. Supposing this upper transition reflects actual dissociation of the third strand from the linear duplex substrate this triplex is comparable in thermodynamic stability to the triplex formed with a dumbbell substrate. Even so, the biphasic melting character of the linear triplex obscures the whole analysis, casting doubt on its absolute reliability. Apparently triplexes formed with a dumbbell substrate offer technical advantages over triplexes formed from linear or hairpin duplex substrates for studies of DNA triplex stability. © 1993 John Wiley & Sons, Inc.
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  • 7
    Electronic Resource
    Electronic Resource
    Weinheim : Wiley-Blackwell
    Electrophoresis 13 (1992), S. 521-528 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Alternative protocols are necessary for the use of polyacrylamide gel electrophoresis in genome scale sequencing and mapping studies. The use of radioisotopes and manual gel reading will have to be replaced with a flexible labeling system that can be detected at levels similar to or better than radioisotopes but allows automated, high-speed detection. Labeling with stable isotopes is such an alternative. These nondecaying isotopes have the potential to be detected in sub-attomole quantities, despite being surrounded by the gel matrix, due to the high selectivity and sensitivity of resonance-ionization spectroscopy coupled with a mass spectrometer. In this study the detection limits of sputter-initiated resonance ionization spectroscopy (SIRIS) are investigated using thin, open-faced polyacrylamide gels supported by plastic. This system allows reproducibility and flexibility in the choice of gel size and buffer system since the gel can be cast, washed free of polymerization by-products, dried, and stored until use. Various concentrations of an Sn-labeled oligomer were run on these gels and loads of 5 femtomoles/mm could be detected on a 240 μm thick gel. Gels as thin as 60 μm lower the detectable concentration loads to 1 femtomole/mm. The limiting factor is tin contamination in the gel which, if reduced, will further increase detection. Polymerase chain reaction (PCR) products can also be labeled and detected using Sn isotopes, which could prove useful in mapping studies. Also presented are techniques which will facilitate resolution of these PCR products on open-faced gels by employing discontinuous buffers systems and DNA mobility modifiers.
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  • 8
  • 9
    Publication Date: 2013-09-26
    Electronic ISSN: 2161-5063
    Topics: Biology , Chemistry and Pharmacology
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  • 10
    Publication Date: 2009-03-17
    Print ISSN: 0743-7463
    Electronic ISSN: 1520-5827
    Topics: Chemistry and Pharmacology
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