Analysis of high‐resolution melting (thermal dispersion) of DNA. Methods

Abstract

SynopsisWe describe the capabilities of a method for obtaining high-resolution melting profiles of short, homogeneous DNA9 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"Chr 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 0.2OC. 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.

Segmentation of DNA sequences into twostate regions and melting fork regions

“…These findings were in accordance with Azbel's prediction of the deviation of a domain's T m from the GC%-based Marmur-Doty prediction [12,10,2,1]. He distinguished five types of subtransition of a melting domain by the number of boundaries they create: (I) nucleation of a bubble, (II) unzipping from an end, (III) internal growth of a bubble, (IV) merging of a bubble and an unzipping end, and (V) merging of two bubbles.…”

“…Four of these correspond to meltmap types of flat regions: top, bottom, upstair, and downstair [15]. The subtransition indicates the most likely melting process of each region [10,23], based on Azbel's distinction according to the change n b in the number of boundaries [12]. The effect on T m is the deviation from the expectation based on GC% content [12].…”

Characterization of Mycobacteriophage I8 and its Unrelatedness to Mycobacteriophages I1, I3 and I5

“…4 (b). The number of subtransitions was less in the I8 DNA profile compared to 2 DNA (Yen & Blake, 1980). This provided further support for the random distribution of bases in 18 DNA.…”

Loop energy in DNA

“…Derivative melting curves were analytically reconstructed from differential curves as previously described. 13 The distribution of (G + The rate of heating was 6 deg . h-', which is approximately threefold slower than necessary to ensure an equilibrium state at all temperatures (see below).…”

“…The variation of D with the residual stacking in loops can be estimated by setting ( N , -N ) J N , equal to (1 -6,), and then calculating N from Eq. (13) with different values of D, while maintaining TAAN(N) = TAAN(Ni). Figure 11 shows a plot of the expected variation in D with the fraction of residual stacking.…”