Modeling of two-dimensional DNA display
📝 Abstract
2D display is a fast and economical way of visualizing polymorphism and comparing genomes, which is based on the separation of DNA fragments in two steps, according first to their size and then to their sequence composition. In this paper, we present an exhaustive study of the numerical issues associated with a model aimed at predicting the final absolute locations of DNA fragments in 2D display experiments. We show that simple expressions for the mobility of DNA fragments in both dimensions allow one to reproduce experimental final absolute locations to better than experimental uncertainties. On the other hand, our simulations also point out that the results of 2D display experiments are not sufficient to determine the best set of parameters for the modeling of fragments separation in the second dimension and that additional detailed measurements of the mobility of a few sequences are necessary to achieve this goal. We hope that this work will help in establishing simulations as a powerful tool to optimize experimental conditions without having to perform a large number of preliminary experiments and to estimate whether 2D DNA display is suited to identify a mutation or a genetic difference that is expected to exist between the genomes of closely related organisms.
💡 Analysis
2D display is a fast and economical way of visualizing polymorphism and comparing genomes, which is based on the separation of DNA fragments in two steps, according first to their size and then to their sequence composition. In this paper, we present an exhaustive study of the numerical issues associated with a model aimed at predicting the final absolute locations of DNA fragments in 2D display experiments. We show that simple expressions for the mobility of DNA fragments in both dimensions allow one to reproduce experimental final absolute locations to better than experimental uncertainties. On the other hand, our simulations also point out that the results of 2D display experiments are not sufficient to determine the best set of parameters for the modeling of fragments separation in the second dimension and that additional detailed measurements of the mobility of a few sequences are necessary to achieve this goal. We hope that this work will help in establishing simulations as a powerful tool to optimize experimental conditions without having to perform a large number of preliminary experiments and to estimate whether 2D DNA display is suited to identify a mutation or a genetic difference that is expected to exist between the genomes of closely related organisms.
📄 Content
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Modeling of two-dimensional DNA display
Ana-Maria FLORESCU (1), Marc JOYEUX (1) and Bénédicte LAFAY (2)
(1) Laboratoire de Spectrométrie Physique (CNRS UMR5588), Université Joseph Fourier Grenoble 1, BP 87, 38402 St Martin d’Hères, France
(2) Laboratoire Ampère (CNRS UMR5005), Université de Lyon, Ecole Centrale de Lyon, 36 avenue Guy de Collongue, 69134 Ecully, France
Corresponding author :
Marc JOYEUX
Laboratoire de Spectrométrie Physique (CNRS UMR5588)
Université Joseph Fourier Grenoble 1
BP 87
38402 St Martin d’Hères
France
Email : Marc.Joyeux@ujf-grenoble.fr
Phone : (+33) 476 51 47 51
Fax : (+33) 476 63 54 95
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Abstract : 2D display is a fast and economical way of visualizing polymorphism and comparing genomes, which is based on the separation of DNA fragments in two steps, according first to their size and then to their sequence composition. In this paper, we present an exhaustive study of the numerical issues associated with a model aimed at predicting the final absolute locations of DNA fragments in 2D display experiments. We show that simple expressions for the mobility of DNA fragments in both dimensions allow one to reproduce experimental final absolute locations to better than experimental uncertainties. On the other hand, our simulations also point out that the results of 2D display experiments are not sufficient to determine the best set of parameters for the modeling of fragments separation in the second dimension and that additional detailed measurements of the mobility of a few sequences are necessary to achieve this goal. We hope that this work will help in establishing simulations as a powerful tool to optimize experimental conditions without having to perform a large number of preliminary experiments and to estimate whether 2D DNA display is suited to identify a mutation or a genetic difference that is expected to exist between the genomes of closely related organisms.
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1 - Introduction
Two-dimensional (2D) DNA display was first described by Fisher and Lerman [1-3]. This is an electrophoresis technique, which consists in separating DNA fragments in two steps, according first to their size and then to their sequence composition. The first step uses traditional slab electrophoresis, for example in agarose or polyacrylamide gels. Collisions between DNA and the gel reduce the mobility of DNA fragments, so that the gel acts as a sieve and the electrophoretic mobility becomes size-dependent, with smaller molecules generally going faster than large ones [4]. In the second dimension, fragments of identical length are separated on the basis of their sequence composition, thanks to a gradient of either temperature (TGGE : temperature gradient gel electrophoresis) or the concentration of a chemical denaturant, e.g., a mixture of urea and formamide (DGGE : denaturing gradient gel electrophoresis), both methods being closely related (see [5,6] and below). The effective volume of denaturated regions being larger than that of double-stranded ones, the mobility of a given fragment decreases as the number of open base pairs increases. Since AT-rich regions melt at lower temperatures than GC-rich ones, GC-rich fragments usually move farther than AT-rich ones.
Although 2D DNA display has recently been applied to the comparison of the genomes of closely related bacteria [7-9], this method is still essentially empirical and simulations have only been used to a very limited extent to plan experiments and interpret results [10-12]. In particular, it has been shown only recently [12] that the final relative positions of DNA fragments in 2D display experiments can be predicted with satisfying precision using a model that combines step-by-step integration of the equation of motion of each fragment and the use of the open source program Meltsim [13] to estimate the number of open base pairs at each step of the DGGE phase. This kind of simulations will therefore
4 certainly develop further, in order to help optimize experimental conditions (denaturing gradient range, electrophoresis duration, etc) without having to perform a large number of tedious preliminary experiments, and to predict whether 2D DNA display is a convenient tool to identify a given mutation or a genetic difference that is expected to exist between the genomes of closely related organisms.
The goal of this paper is to extend the results presented in [12] along several lines. First, these results were obtained by modifying one parameter in existing formulae for the mobility of the DNA fragments during each phase of the 2D display process [5,14,15]. The point is that the formula, which was used to estimate the mobility of the fragments during electrophoresis along the first dimension, is rather complex and involves many parameters [14,15]. It is therefore not ideally suited for fitting purposes. We will show that the corresponding procedure c
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