Melting curves nucleic acids

A plot of the optical absorbance at 260 nm (the wavelength of maximum light absorption by nucleic acids) versus temperature is known as a melting curve (Fig. 5-45). The absorbance is lower, by up to 40%, for native than for denatured nucleic acids. This hypochromic effect (Chapter 23) is a result of the interaction between the closely stacked bases in the helices of the native molecules. The melting temperature Tm is taken as the midpoint of the increase in absorbance (Fig. 5-45). As the percentage of G + C increases, the nucleic acid becomes more stable toward denaturation because of the three hydrogen bonds in each GC pair. Tm increases almost linearly with increases in the G + C content. In the "standard" citrate buffer (0.15 M NaCl + 0.015 M sodium citrate, pH 7.0) Eq. 5-22 holds. The exact numerical relationship depends strongly upon the ionic composition and pH of the medium.37 72 552 553... 

Whereas proteins have their low energy absorption band at 280 nm, polynucleotides typically have maxima at 260 nm (38,500 cm ). A phenomenon of particular importance in the study of nucleic acids is the hypochromic effect. In a denatured polynucleotide the absorption is approximately the sum of that of the individual components. However, when a double helical structure is formed and the bases are stacked together, there is as much as a 34% depression in the absorbance at 260 nm. This provides the basis for optical measurement of DNA melting curves (Fig. 5-45).45,86 The physical basis for the hypochromic effect is found in dipole-dipole interactions between the closely stacked base pairs.7,86,87... 

To determine whether the nucleic acid is single-stranded or double-stranded, it could be heated. A sharp increase in absorbance at 260 nm would indicate a double-stranded RNA or DNA a broader melting curve would suggest a single-stranded nu-... 

The melting transition of the daunomycin poly(dA-dT) complex can also be monitored at the nucleic acid resonance line widths and the data for the adenosine H-8 resonance are plotted in Figure 28. The resonance is very broad at temperatures below the melting transition of the complexes (dashed curves in Figure 28) indicative of stiffening of the synthetic DNA by the bound anthracycline ring. 

Tubular membrane tension/mucosal DNA thermal melting point midpoint of thermal denaturation curve Transmembrane Trimethylamine oxide Transmembrane domain Trimethylsilyl thimersol Tobacco mosaic virus Treose nucleic acid 5-thio-2-nitrobenzoate... 

Figure 37-19 Melting curve of double-helical nucleic acid. (Modified with permission from Piper MA, Unger ER Nucleic Add Probes A Primer for Potho/og/sts. Chicago, ASCP Press, 1989.)...

Tm. The melting point for a double-stranded nucleic acid. Technically, this is defined as the temperature at which 50% of the strands are in double-stranded form and 50% are single-stranded, i.e., midway in the melting curve. A primer has a specific Tm because it is assumed that it will find an opposite strand of appropriate character. 

R.A.J. Darby, M. Sollogoub, C. McKeen, L. Brown, A. Risitano, N. Brown, C. Barton, T. Brown and K.R. Fox, High throughput measurement of duplex, triplex and quadruplex melting curves using molecular beacons and a LightCycler, Nucleic Acids Res., 2002, 30(9), 39. 

Wittwer, C. T. Gundry, C. High resolution genotyping of nucleic acid polymorphisms using melting curve analysis. Eur. Pat. Appl. EP 1362928, 2003 Chem. Abstr. 2003,139, 376184. 

Choice of a buffer with an appropriate pK to give sufficient buffer capacity at the desired pH is important to prevent changes in pH as a result of a nucleic acid structural transition. Since nucleic add concentrations for UV melting studies are routinely 10" to 10" m and most nucleic add structural transitions do not involve the absorption or release of very many equivalents of add (C -GC triple helices are a notable exception), a buffer capadty of greater than 1 mM is usually sufficient to prevent large fluctuations in pH during a melting curve. For example, we and others have observed that the adenine N1 of A -C... 

The strength of noncovalent interactions of nucleic acid complexes is routinely determined by their temperature-dependent melting behavior in solution where the fraction of intact complex (oc) is determined as a function of the temperature of the solution. UV absorption, fluorescence, or circular dichroism is usually employed as the means of detection of biomolecular interactions in this type of experiments. A transition enthalpy can be obtained from the slope of the melting curve as described by Marky and Breslauer and given in Eq. (15.7), where n is the molecularity of the association reaction (e.g., n = 2 for dimerization) and Tm is the melting temperature, normally defined for a = 50%. 

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