Anal. Chem. 1995,67,784-786
Rapid Pulsed Field Capillary Electrophoretic Separation of Megabase Nucleic Acids Yongseong Kim and Michael D. Morris* University of Michigan, Department of Chemistry, Ann Arbor, Michigan 48109- 1055
Pulsed field capillary electrophoresisin ultradilute solutions of sieving polymers has been used to separate nucleic acid fragments as long as 1.6 million base pairs. Hydroxyethyl cellulose solutions are used for separations in the size range 8000-50 000 base pairs. Longer chain nucleic acids are separated in mixed hydroxyethyl cellulose/poly(ethylene oxide) solutions. Separations are rapid (12-13 min). It is difficult to separate double-stranded (ds) DNA fragments longer than about 20-30 kbp (thousand base pairs) by constant electric field (dc) capillary gel electrophoresis (CGE). Only partial resolution of fragments as long as 48 kbp has been dem~nstrated.l-~ Above about 50 kbp fragments comigrate without separation. To improve CGE separations, several laboratories have proposed the use of pulsed field capillary gel electrophoresis (PFCGE).5-7 The technique is derived from agarose slab gel pulsed field electrophoresis, which extends classical nucleic acid separation from a size limit of about 20 kbp to at least 10 Mbp (mega base pairs). The initial reports on PFCGE were aimed mostly at technique development and described applications to separation of relatively short ds-DNA fragments (0.5-23 kbp), where only modest increases in resolution and decreases in separation time were expected or observed. In this work, several important experimental techniques were developed, including the initial application to linear polyacrylamide sieving buffer^,^ sinusoidal modulation of the driving voltage! time-delayed field inver~ion,~ and random pulse duration field inversiong in ultradilute polymer solutions7 as well as in more conventional buffer^.^^^^^^^ Although most reports have been to nucleic acid separations, PFCGE has been shown to improve resolution in polysaccharide separations as well.lo The use of ultradilute linear polymers as matrices for nucleic acid capillary electrophoresis was recently proposed by Viovy and Duke” and demonstrated by Grossmann and Soane12and Barron and co-workers’3 to yield rapid separations of moderately short (1) Sudor, J.; Foret, F.; Bocek, P. Electrophoresis 1991, 12,1056-1058. (2) Bocek, P.;Chrambach, A Electrophoresis 1992,13,31-34. (3) Storage, M.: Lagu, M. Anal. Chem. 1991,63,1233-1236. (4) Guszczynski,T.; Pulyaeva, H.; Gamer, M. M.; Chrambach, A Electrophoresis 1993, 14,523-530. (5) Heiger, D. N.; Cohen, A S.; Karger, B. L.]. Chromatogr. 1990,516,3348. (6) Sudor, J.; Novotny, M.V. Anal. Chem. 1994,66, 2446-2450. (7) Kim, Y.;Moms, M. D. Anal. Chem. 1994,66, 3081-3085. (8) Demana, T.;Lanan, M.; Moms, M. D.Anal. Chem. 1991,63,2795-2797. (9) Navin, M.J.; Rapp, T. L.: Moms, M. D.Anal. Chem. 1994, 66, 11791182. (10) Sudor, J.: Novotny, M. V. Proc. Natl. Acad. Sci. U.S.A. 1993,90,94519455. (11) Vvoy, J. L.; Duke, T. Electrophoresis 1993,14,322-329.
784 Analytical Chemistry, Vol. 67, No. 5,March 1, 7995
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