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  • 1
    Digitale Medien
    Digitale Medien
    New York : Wiley-Blackwell
    Biopolymers 19 (1980), S. 681-700 
    ISSN: 0006-3525
    Schlagwort(e): Chemistry ; Polymer and Materials Science
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Chemie und Pharmazie
    Notizen: We describe the capabilities of a method for obtaining high-resolution melting profiles of short, homogeneous DNAs using a thermo-differential absorbance technique. The absorbance difference of two identical DNA solutions, raised linearly in temperature and maintained at a constant temperature difference, is monitored using a double-beam spectrophotometer. A specially constructed temperature controller and cell holder enable the temperature of the DNA samples to be controlled and monitored directly. A heating rate of 6.75°C/hr has been found to give reproducible results at ionic strengths 〉 0.01M. A method of reconstructing the true derivative from experimental data using a Taylor series expansion is described and shown to work well when the difference in temperature between samples is in the range of 0.2°C. Reconstructed derivative profiles are further analyzed by deconvolution into distinct Gaussian components. The melting profile of PM-2 DNA is shown to consist of 14 components, while the much longer lambda DNA yields 55. Related techniques such as data management and analysis for the fractional G·C content of specific domains are also described.
    Zusätzliches Material: 7 Ill.
    Materialart: Digitale Medien
    Bibliothek Standort Signatur Band/Heft/Jahr Verfügbarkeit
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  • 2
    Digitale Medien
    Digitale Medien
    New York : Wiley-Blackwell
    Biopolymers 20 (1981), S. 1161-1181 
    ISSN: 0006-3525
    Schlagwort(e): Chemistry ; Polymer and Materials Science
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Chemie und Pharmazie
    Notizen: The thermal dissociation of DNA by five distinct local processes as outlined by Azbel [(1980) Biopolymers 19, 61-80] is treated as a collection of two-state substransitions. With the traditional expression for loop entropy and the assumption of a small, positive loop-initiation enthalpy, explicit relationships between thermodynamic parameters are derived for all five processes. The effect of each process on subtransitional width, unit enthalpy, and melting temperature is estimated; the latter being in excellent agreement with that predicted by Azbel. Criteria proposed for the deconvolution of the oscillatory high-resolution melting profiles exhibited by short, homogeneous DNAs [Yen & Blake (1980) Biopolymers 19, 681-700] have been applied to profiles of SV40 fragment [Gabbarro-Arpa et al. (1979) Nature 280, 515-517] and φx174-RF DNA at two ionic strengths [Vizard et al. (1978) 275, 250-251] and indicate that dissociation of the strands proceeds as a series of local two-state subtransitions of domains 200-300 base pairs in length. Although the obvious overlap of some subtransitions raises questions about resolution, most sections are very well resolved with the minimum number of subtransitions and yield values for the apparent van't Hoff enthalpy in excellent agreement with those expected by calorimetric measurements. The flexibility of such an approach, where the enthalpy is recognized explicitly as an adjustable parameter, is especially suited for the analysis of profiles from unkown DNA sequences, and for the evaluation of extraordinary variations in free energy, such as that associated with loop formation or that resulting from variations in the linear charge density at low ionic strengths.
    Zusätzliches Material: 9 Ill.
    Materialart: Digitale Medien
    Bibliothek Standort Signatur Band/Heft/Jahr Verfügbarkeit
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