Mobility of a DNA fragment

The electrophoretic mobility shift assay (EMSA), also called the gel-shift or band-shift assay, is more useful than the footprinting assay for quantitative analysis of DNA-binding proteins. In general, the electrophoretic mobility of a DNA fragment is reduced when it is complexed to protein, causing a shift in the location of the fragment band. This assay can be used to detect a transcription factor in protein... 

The fifth step was to correct for the different effects of the dyes on the electrophoretic mobility of the DNA fragments. This was accomplished by making use of a property of the DNA fragment mobilities which is illustrated in Figure 8. In this figure the number in the DNA sequence of the base to which each peak corresponds is plotted versus the data point number (which is proportional to time since the time interval between data points is constant) at which the peak occurs. The data set used here is the NBD channel of a typical four dye data set. Also shown is the line obtained when these data are fit by least-squares analysis to a linear function. The linear time dependence shown here is obtained when the gel electrophoresis is performed under constant current conditions. 

S. A. Allison and S. Mazur, Modeling the free solution electrophoretic mobility of short DNA fragments. Biopolymers, 46,359-373 (1998). 

Mechanistic Ideas. The ordinary-extraordinary transition has also been observed in solutions of dinucleosomal DNA fragments (350 bp) by Schmitz and Lu (12.). Fast and slow relaxation times have been observed as functions of polymer concentration in solutions of single-stranded poly(adenylic acid) (13 14), but these experiments were conducted at relatively high salt and are interpreted as a transition between dilute and semidilute regimes. The ordinary-extraordinary transition has also been observed in low-salt solutions of poly(L-lysine) (15). and poly(styrene sulfonate) (16,17). In poly(L-lysine), which is the best-studied case, the transition is detected only by QLS, which measures the mutual diffusion coefficient. The tracer diffusion coefficient (12), electrical conductivity (12.) / electrophoretic mobility (18.20.21) and intrinsic viscosity (22) do not show the same profound change. It appears that the transition is a manifestation of collective particle dynamics mediated by long-range forces but the mechanistic details of the phenomenon are quite obscure. 

Fig. 6. Plot of log(mobility) of a mixture of DNA restriction fragments against logfnumber of base pairs). Separation conditions L=40-47 cm buffer 0.1 M TBE, pH 8.3 with 3% T, 0% C LPA various field strengths and temperatures...

Since DNA fragments from the PCR typically are contained in a high salt matrix, their mobility will vary depending on sample salt concentration. Thus, proper identification of these DNA fragments requires the use of an internal standard to normalize analyte velocity. This practice corrects for variance in fragment mobility due to sample matrix differences (i.e., salt content). These internal standards are included for size determination (in bp) as well as a reference for migration time. Candidates for such internal standards include the primer or primer-dimer peaks, since both components are already present in the PCR mixture alternatively, one or more coinjected standard DNA peak s may be chosen. If any of these fragments are to serve as the internal standard, they must be separated from one another and any PCR product, a precondition that is not easily met when the size of the PCR product is below 60 bp. 

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