Mobility, diffusion and dispersion of single‐stranded DNA in sequencing gels

Electrophoresis of single‐stranded DNA in denaturing polyacrylamide gels is presently a standard procedure for the sequencing of DNA fragments. A thorough understanding of the factors that determine the resolution of DNA fractionated in polyacrylamide gels is necessary to optimize the performance of DNA sequencers. Significant research on the mobility of double‐stranded (ds)DNA molecules in agarose and polyacrylamide gels has been performed, and the phenomenon of band broadening of single‐stranded (ss)DNA fragments in DNA sequencing gels has received attention only recently. In this paper, we present a detailed study of mobility, diffusion and dispersion of ssDNA in sequencing gels as a function of molecular size, gel concentration and electric field strength. DNA mobility is shown to be essentially independent of electric field in the range of 0‐60 V/cm. The band broadening is greatly enhanced in the presence of an electric field and the dispersion coefficient (DE) can be an order of magnitude higher than the field‐free diffusion coefficient. The measured migration parameters approximately follow the predictions of the biased reptation including fluctuations (BRF) theory. However, deviations due to nonidealities of the separation conditions are observed. The measured migration parameters can be used to optimize the performance of separation systems.

[1]  A. Kloosterman,et al.  Comparison of resolution of double-stranded and single-stranded DNA in capillary electrophoresis , 1997 .

[2]  Lloyd M. Smith,et al.  Analysis of resolution in DNA sequencing by capillary gel electrophoresis , 1993 .

[3]  G. Weill,et al.  Brownian diffusion and electrophoretic transport of double-stranded DNA in agarose gels , 1999 .

[4]  T. Duke,et al.  Electrophoretic mobility of DNA in gels. II. Systematic experimental study in agarose gels , 1994 .

[5]  M. Sepaniak,et al.  Examination of band dispersion during size-selective capillary electrophoresis separations of DNA fragments. , 1997, Journal of chromatography. B, Biomedical sciences and applications.

[6]  Gary W. Slater,et al.  Entropic Trapping of DNA During Gel Electrophoresis: Effect of Field Intensity and Gel Concentration , 1997 .

[7]  G. Slater,et al.  Theory of band broadening for DNA gel electrophoresis and sequencing , 1993, Electrophoresis.

[8]  Jean Sturm,et al.  Persistence Length of Single-Stranded DNA , 1997 .

[9]  B. Zimm,et al.  Theory of gel electrophoresis of DNA , 1985, Biopolymers.

[10]  Christoph Heller.  Separation of double‐stranded and single‐stranded DNA in polymer solutions: I. Mobility and separation mechanism , 1999, Electrophoresis.

[11]  S. Edwards,et al.  The Theory of Polymer Dynamics , 1986 .

[12]  Analysis of limiting factors of DNA band separation by a DNA sequencer using fluorescence detection , 1991, Electrophoresis.

[13]  G. Slater,et al.  On the reptation theory of gel electrophoresis , 1986 .

[14]  L. Ziaugra,et al.  DNA sequencing on microfabricated electrophoretic devices. , 1998, Analytical chemistry.

[15]  A. G. Ogston,et al.  The spaces in a uniform random suspension of fibres , 1958 .

[16]  H. Frisch,et al.  Why does the electrophoretic mobility of DNA in gels vary with the length of the molecule? , 1982, Biopolymers.

[17]  C. Desruisseaux,et al.  The gel edge electric field gradients in denaturing polyacrylamide gel electrophoresis , 1998, Electrophoresis.

[18]  D. Schwartz,et al.  Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis , 1984, Cell.

[19]  Charles Anderson,et al.  Importance of oligoelectrolyte end effects for the thermodynamics of conformational transitions of nucleic acid oligomers: A grand canonical Monte Carlo analysis , 1991, Biopolymers.

[20]  R A Mathies,et al.  Optimization of high-speed DNA sequencing on microfabricated capillary electrophoresis channels. , 1999, Analytical chemistry.

[21]  The relative contributions of dispersion and diffusion to band spreading (resolution) in gel electrophoresis , 1996, Electrophoresis.

[22]  G. Drouin,et al.  Gel electrophoretic mobility of single‐stranded DNA: The two reptation field‐dependent factors , 2000, Electrophoresis.

[23]  G. Weiss,et al.  Interpretation of electrophoretic band shapes by a partition chromatographic model , 1996, Electrophoresis.

[24]  T. Duke,et al.  Electrophoretic mobility of DNA in gels. I. New biased reptation theory including fluctuations , 1994 .

[25]  Christoph Heller.  Separation of double‐stranded and single‐stranded DNA in polymer solutions: II. Separation, peak width and resolution , 1999, Electrophoresis.

[26]  R. Larson.  The Structure and Rheology of Complex Fluids , 1998 .

[27]  J. Viovy.  Electrophoresis of DNA and other polyelectrolytes: Physical mechanisms , 2000 .

[28]  Gary W. Slater,et al.  Theory of DNA sequencing using free-solution electrophoresis of protein-DNA complexes , 1994 .

[29]  G. Drouin,et al.  On the limits of near‐equilibrium DNA gel electrophoretic sequencing , 1993, Electrophoresis.

[30]  A. Pluen,et al.  Band broadening in gel electrophoresis: scaling laws for the dispersion coefficient measured by FRAP. , 1998, Biopolymers.

[31]  A. Pluen,et al.  Migration of single‐stranded DNA in polyacrylamide gels during electrophoresis , 1998, Electrophoresis.

[32]  G. Slater,et al.  Ogston gel electrophoretic sieving: How is the fractional volume available to a particle related to its mobility and diffusion coefficient(s)? , 1995, Electrophoresis.

[33]  N. Stellwagen,et al.  Estimation of polyacrylamide gel pore size from Ferguson plots of normal and anomalously migrating DNA fragments. I. Gels containing 3 % N, N′‐methylenebisacrylamide , 1991, Electrophoresis.