Salt dependence and thermodynamic interpretation of the thermal denaturation of small DNA restriction fragments

Abstract

The influence of cation concentration on the thermal denaturation of DNA restriction fragments from the E. coli lac regulatory region and from pVH51, ranging in size from 43- to 880- bp, is described. Upon increasing the ionic strength, the melting transitions broaden in a cooperative manner at salt concentrations characteristic for the specific fragment. For three fragments studied in detail, the salt concentration dependence at the midpoint varied between 0.03 and 0.19 M Na+. Along with the broadening, the melting transitions become more symmetrical. This result is discussed with respect to the irreversibility of melting transitions at low ionic strength. After a cooperative broadening, the shape of the melting curves remains constant up to salt concentrations of 0.5 M Na+. The dTM/dlog[Na+] values for three fragments fall between 15.7 and 16.7. An easily applicable approximation of the van't Hoff equation is used to evaluate the enthalpies of 13 transitions arising from the denaturation of 43 to 600 bp. The results of this analysis are compared to calculations of the expected enthalpies based on calorimetric measurements. The TMs of most transitions were directly related to the base composition, but several deviations from the predicted behavior were observed. The possible influences of fragment length and sequence on the thermal stability are discussed. The experimental and mathematical procedure to resolve a thermal denaturation transition with a width f 0.17 +/- 0.01 degrees and its distinction from another preceeding transition only approximately 0.15 degrees away in an 880-bp Hae III fragment from pVH51 is described. This transition is about half as wide as the smallest one reported to date.

Correlation of thermodynamic and genetic properties in the Tn10 encoded TET gene control region

“…It is noteworthy that the melting enthalpies of all transitions support the area analysis of the melting experiments with the exception of the second sharp transition in the 1450-bp DNA [16,19]. It may thus be, that this transition is the result of two melting processes separated only by a small difference in TM.…”

“…sitions resulting from TET gene sequences. As mentioned above step 2 may actually consists of two individual reactions closely spaced on the temperature scale [16].…”

“…A second thermal bath was used to maintain the temperature of the photomultiplyer housing at 400C. The other processing of the data was as described [16]. The melting curves were plotted using a HP 9862A plotter from Hewlett Pakkard.…”

Solution composition and thermal denaturation for the production of single-stranded PCR amplicons: Piperidine-induced destabilization of the DNA duplex?

“…At pH values below the pKa of piperidine, the excess of protonated piperidine acts as a cation and electrostatically binds to the phosphate backbone neutralizing the repulsive negative charges and thus creating an effect similar to peptide nucleic acid (PNA) duplex stability where Coulombic repulsions have been eliminated [47]. This effect is analogous to the Na ϩ cation binding to the negatively charged phosphate backbone stabilizing the duplex by reducing the electrostatic repulsion [48,49]. At pH values above the suggested pKa of piperidine, the excess of neutral piperidine can hydrogen bond with the nucleobases destabilizing the double helix analogous to the denaturant formamide [50].…”

Development of a Molecular Platform for GMO Detection in Food and Feed on the Basis of “Combinatory qPCR” Technology

“…The melting temperature (T m ) of a DNA sequence is dependent on a large number of factors, among which the ionic conditions in the sample solution, the DNA nature (sequence, secondary structure, etc.) and the starting concentration of the DNA molecule (Hillen et al, 1981;Rouzina & Bloomfield, 2001). Moreover different qPCR instruments tend to measure slightly different values for a given amplicon (due to differences in heating block control, mathematical integration, extrapolation, etc.).…”