Determining the electrophoretic mobility and translational diffusion coefficients of DNA molecules in free solution

Abstract

The free solution mobility of DNA molecules of different molecular weights, the sequence dependence of the mobility, and the diffusion coefficients of small single- and double-stranded DNA (ss- and dsDNA) molecules can be measured accurately by capillary zone electrophoresis, using coated capillaries to minimize the electroosmotic flow (EOF) of the solvent. Very small differences in mobility between various analytes can be quantified if a mobility marker is used to correct for small differences in EOF between successive experiments. Using mobility markers, the molecular weight at which the free solution mobility of dsDNA becomes independent of molecular weight is found to be approximately 170 bp in 40 mM Tris-acetate-EDTA buffer. A DNA fragment containing 170 bp has a contour length of approximately 58 nm, close to the persistence length of DNA under these buffer conditions. Hence, the approach of the free solution mobility of DNA to a plateau value may be associated with the transition from a rod-like to a coil-like conformation in solution. Markers have also been used to determine that the free solution mobilities of ss- and dsDNA oligomers are sequence-dependent. Double-stranded 20-bp oligomers containing runs of three or more adenine residues in a row (A-tracts) migrate somewhat more slowly than 20-mers without A-tracts, suggesting that somewhat larger numbers of counterions are condensed in the ion atmospheres of A-tract DNAs, decreasing their net effective charge. Single-stranded 20-mers with symmetric sequences migrate approximately 1% faster than their double-stranded counterparts, and faster than single-stranded 20-mers containing A(5)- or T(5)-tracts. Interestingly, the average mobility of two complementary single-stranded 20-mers is equal to the mobility of the double-stranded oligomer formed upon annealing. Finally, the stopped migration method has been used to measure the diffusion coefficients of single- and double-stranded oligomers. The diffusion coefficients of ssDNA oligomers containing 20 nucleotides are approximately 50% larger than those of double-stranded DNA oligomers of the same size, reflecting the greater flexibility of ssDNA molecules. The methods used to carry out these experiments are also described in detail.

On the importance of hydrodynamic interactions in polyelectrolyte electrophoresis

“…This approach was successfully applied in simulations to determine the electrophoretic mobility of charged colloids [18,19]. In an earlier publication this year [12], we compared the simulation results to experimental data for short PSS obtained by two different experimental methods, namely, capillary electrophoresis [8,20] and pulsed field gradient or electrophoretic NMR [21,22,23,24] 1 . In Figure 1 [ 11,25,26,27].…”

The role of DNA diffusion in solid phase polymerase chain reaction with gel-immobilized primers in planar and capillary microarray format

“…It is interesting to compare this value to earlier published data for diffusion coefficient D using the formula r m =2͑D ·1 s͒ 1/2 . For 20-base oligonucleotide at 20°C the D = 1.52ϫ 10 −6 cm 2 / s. 28 The empirical relationship D ϰ N −0.67 for N-base ds-DNA ͑Ref. 29͒ was successfully applied to single stranded oligonuclotides.…”

Efficient simulation of semiflexible polymers

“…The diffusion coefficient of small dsDNA segments has been studied using various techniques, such as capillary electrophoresis [31,32], dynamic light scattering [33], NMR [34], and fluorescent recovery after photobleaching [35]. Of these, the first three converge on the value D ≈ 1.07 × …”