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A theoretical study of the possible use of electroosmotic flow to extend the read length of DNA sequencing by end‐labeled free solution electrophoresis
Author(s) -
McCormick Laurette C.,
Slater Gary W.
Publication year - 2006
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200500573
Subject(s) - electrophoresis , capillary electrophoresis , dna , chemistry , flow (mathematics) , free flow electrophoresis , limiting , molecule , chromatography , analytical chemistry (journal) , biological system , chemical physics , physics , mechanics , gel electrophoresis of proteins , polyacrylamide gel electrophoresis , biology , mechanical engineering , biochemistry , organic chemistry , engineering , enzyme
End‐labeled free solution electrophoresis (ELFSE) provides a means of separating DNA with free‐solution CE, eliminating the need for gels and polymer solutions which increase the run time and can be difficult to load into a capillary. In free‐solution electrophoresis, DNA is normally free‐draining and all fragments reach the detector at the same time, whereas ELFSE uses an uncharged label molecule attached to each DNA fragment in order to render the electrophoretic mobility size‐dependent. With ELFSE, however, the larger molecules are not separated enough (limiting the read length in the case of ssDNA sequencing) while the smaller ones are overseparated; the larger ones are too fast while the shorter ones are too slow, which is the opposite of traditional gel‐based methods. In this article, we show how an EOF could be used to overcome these problems and extend the DNA sequencing read length of ELFSE. This counterflow would allow the larger, previously unresolved molecules more time to separate and thereby increase the read length. Through our theoretical investigation, we predict that an EOF mobility of approximately the same magnitude as that of unlabeled DNA would provide the best results for the regime where all molecules move in the same direction. Even better resolution would be possible for smaller values of EOF which allow different directions of migration; however, the migration times then would become too large. The flow would need to be well controlled since the gain in read length decreases as the magnitude of the counterflow increases; an EOF mobility double that of unlabeled DNA would no longer increase the read length, although ELFSE would still benefit from a reduction in migration time.