Application of higher derivative techniques to analysis of high-resolution thermal denaturation profiles of reassociated repetitive DNA.

Abstract

ABSTRACr We have analyzed high-resolution denaturation profiles of reassociated repetitive DNA sequences by using a combination of higher derivative analysis and curve-fitting techniques. Procedures originally used for resolution of components in complex absorption spectra were found to be applicable to high-resolution analysis of melting profiles of reassociated repetitive DNA sequences from pea DNA. Under conditions that eliminate the base composition effect on thermal stability (24 M tetraethylamonium chloride), such an analysis can distinguish "thermal classes" of repetitive DNA duplexes exhibiting different amounts of base pair mismatch. Oly a singl thermal class is observed in reassociated Eshberichia colt DNA whereas at least five classes can be reproducibly distinguished in pea and mung bean DNAs.High-resolution thermal denaturation profiles have been successfully used in the analysis of simple prokaryotic-genomes such as those of bacteriophages OX174, T4, and X (1, 2). Firstderivative analysis of such melting curves reveals numerous discrete components that are suggestive of gene-sized sequences with thermal stabilities reflecting their characteristic G-C content (2, 3).Thermal denaturation profiles have also been used to characterize reassociated repetitive DNA sequences from eukaryotes (e.g., refs. 4-7). Reassociated repetitive eukaryotic DNA typically melts over a much broader range of temperature, and with a lower average thermal stability, than does the corresponding native DNA. It is generally accepted that such profiles reflect the presence of many imperfectly paired duplexes resulting from reactions between divergent members of repetitive sequence families (5). The reduction in average thermal stability (tm) relative to native DNA can provide an estimate of the average base pair mismatch in a given population of duplexes in vitro (5)

Repeat sequence interspersion in coding DNA of peas does not reflect that in total pea DNA

“…Most pea repeat families show extensive sequence divergence [9,16,17,20]. We have chosen to compare the thermal stability of the noncontiguous fraction with that of the most slowly reassociating fraction of 300NT pea fragments.…”

“…Most of the pea genome reassociates to form extensively mismatched duplexes [9,16,20]. As illustrated in Figure 3, when a slowly reassociating fraction is obtained from 300NT rather than 1300NT fragments it also produces duplexes with a low modal stability.…”

“…Samples were reassociated as described above, diluted into a large excess of 0.12M PB and preheated to various temperatures using a thermal gradient block [5] prior to fractionation on hydroxylapatite. A cubic spline computer program was used to fit a smooth curve to the resulting data and first derivatives of the spline-fit curves computed as described [9,17].…”

Numerical analysis of density gradient centrifugation profiles from eukaryotic DNA

“…Either the Medgyessy technique [14,15] or the higher derivative technique [16,17], which originally was developed for the analysis of absorption spectra [18,19], can be applied. Several mathematical procedures to solve this problem have been proposed.…”

“…Symposium on Analytical Ultracentrifugation", Marburg, FRG, February [16][17]1989. Symposium on Analytical Ultracentrifugation", Marburg, FRG, February [16][17]1989.…”