Free-solution electrophoresis of DNA modified with drag-tags at both ends.
In end-labeled free-solution electrophoresis (ELFSE), DNA molecules are labeled with a frictional modifier or "drag-tag", allowing their size-based electrophoretic separation in free solution. Among the interesting observations from early work with dsDNA using streptavidin as a drag-tag was that the drag induced by including a streptavidin label at both ends was significantly more than double that from a single streptavidin (Heller, C. et al.., J. Chromatogr. A 1998, 806, 113-121). This finding was assumed to be in error, and subsequent work focused on experiments in which only a single drag-tag is appended to one end of the DNA molecule. Recent theoretical work (McCormick, L. C., Slater, G. W., Electrophoresis 2005, 26, 1659-1667) has examined the contribution of end-effects to the free-solution electrophoretic mobility of charged-uncharged polymer conjugates, reopening the question of enhanced drag from placing a drag-tag at both ends. In this study, this effect is investigated experimentally, using custom-synthesized ssDNA oligonucleotides allowing the attachment of drag-tags to one or both ends, as well as dsDNA PCR products generated with primers appropriate for the attachment of drag-tags at one or both ends. A range of sizes of drag-tags are used, including synthetic polypeptoid drag-tags as well as genetically engineered protein polymer drag-tags. The enhanced drag arising from labeling both ends has been confirmed, with 6-9% additional drag for the ssDNA and 10-23% additional drag for the dsDNA arising from labeling both ends than would be expected from simply doubling the size of the drag-tag at one end. The experimental results for ssDNA labeled at both ends are compared to the predictions of the recent theory of end-effects, with reasonably good quantitative agreement. These experimental findings demonstrate the feasibility of enhancing ELFSE separations by labeling both ends of the DNA molecule, leading to greater resolving power and a wider range of applications for this technique.
Meagher
Robert J
RJ
Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL 60208, USA.
McCormick
Laurette C
LC
Haynes
Russell D
RD
Won
Jong-In
JI
Lin
Jennifer S
JS
Slater
Gary W
GW
Barron
Annelise E
AE
D004247:DNA / Q000032:analysis; D004277:DNA, Single-Stranded / Q000032:analysis*; D019075:Electrophoresis, Capillary / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*; D012996:Solutions
D004277:DNA, Single-Stranded; D012996:Solutions; D004247:DNA