The molecular end effect and its critical impact on the behavior of charged-uncharged polymer conjugates during free-solution electrophoresis

Abstract

Recently two novel techniques using free-solution electrophoresis to separate charged-uncharged polymer conjugates have proven successful: end-labeled free-solution electrophoresis (ELFSE) for DNA sequencing, and free-solution conjugate electrophoresis (FSCE) for molar mass profiling of uncharged polymers. The approach taken to analyze the experimental data was an extension of the theory of Long and co-workers (Long, D., Dobrynin, A. V., Rubinstein, M., Ajdari, A., J. Chem. Phys. 1998, 108, 1234-1244) for the electrophoresis of molecules with varying charge distributions. This theory also predicts that the ends of the polymers play a large role in determining the polymer's overall mobility; however, this aspect of the theory was neglected in previous work. Until now this "end effect" has, to the knowledge of the authors, not been recognized in experimental data. Through a careful investigation of the predicted end effect and a reanalysis of the experimental data, we demonstrate that indeed this effect critically impacts on the behavior of charged-uncharged polymer conjugates during electrophoresis. This work indicates that not only does the end effect need to be taken into account to avoid significant errors in data analysis, but also it provides novel system optimization approaches.

Molecular deformation and free‐solution electrophoresis of DNA‐uncharged polymer conjugates at high field strengths: Theoretical predictions. Part 1: Hydrodynamic segregation

“…We have neglected the end effects predicted by Long et al [17], for which we amended our theory specifically for ELFSE in a previous paper [18]. These effects, confirmed experimentally in [19], result from monomers near the end of the ELFSE conjugate receiving a higher weighting in the average determining the mobility.…”

“…We note that the approach taken assumes that the linearization procedure of Long and coworkers, where the electric and hydrodynamic forces are considered independently and then superposed, is still a valid approximation for high field strengths (and/or large labels). We also note that, for simplicity, we are neglecting the end effects that were predicted by the theory of Long et al [17] and subsequently examined and confirmed for the case of ELFSE [18,19].…”

Free‐solution electrophoresis of DNA modified with drag‐tags at both ends

“…(1) was recently re-examined theoretically [26]. Whereas previous theory assumed that each monomer unit (after rescaling the uncharged monomers by a 1 ) contributes equally to the electrophoretic mobility of the composite molecule, more recent theory has taken into account end-effects originally described by Long et al [27].…”

“…The normalized weighting function c(n/N) of a Gaussian polymer chain was found in [26] to be well represented by the following function:…”

Branched polymeric labels used as drag-tags in free-solution electrophoresis of ssDNA

“…Since the theory of ELFSE is rather well documented [9,10], we will use the classical model (described in Section 2) to analyze the recent experimental data (presented in Section 3) with linear and branched labels [8]. In order to compute the effective hydrodynamic radius of the various branched labels, we will first use the freely jointed chain (FJC) model and the equations derived by Teraoka [11] for branched FJC polymers.…”