Electrophoresis zyxwvutsrqponmlkjih 1995, zyxwvutsrqponm 16, zyxwvutsrqponm 377-388 Sequence-dependent migration of dsDNA in CE 377 Jan Berka Yvan F. Pariat Odilo Miiller Kirstin Hebenbrock David N. Heiger* FrantiSek Foret Barry L. Karger Barnett Institute and Department of Chemistry Northeastern University Boston, MA Sequence dependent migration behavior of double-stranded DNA in capillary electrophoresis Capillary electrophoresis (CE) with a replaceable linear polyacrylamide (LPA) sieving matrix was used to examine sequence-dependent migration of double- stranded DNA fragments. It has been found that DNA conformational effects were significant under high electric field separations, especially using high resolution matrices. Compared to linear DNA-ladder standards, both anoma- lously slow and rapid DNA fragments were observed, with the degree of ano- malous migration depending on the electric field strength, polymer concentra- tion, column temperature, and background electrolyte (denaturants, sodium and magnesium ions, DNA-intercalating dyes). By selecting a combination of electrophoretic conditions (e.g. 3 zyxwv O/o T LPA, elevated capillary temperature, lower electric field strength and addition of DNA intercalating dyes), mol- ecular weight dependent separations were closely restored**. 1 Introduction A basic prerequisite of electrophoretic separation of nuc- leic acids using gel sieving is accuracy in size determina- tion. As electrophoretic mobilities of DNA species in gel matrices are molecular weight and conformation depen- dent, the final separation pattern may not be in the order of increasing base pair. Indeed, DNA conformation can be examined by electrophoretic behavior [I]. The impor- tant effect of DNA conformation polymorphism on mobility in capillary electrophoresis (CE) is the subject of this work. Structural polymorphism has been shown to be an inher- ent feature of DNA and a source of function variability. One of the most biologically relevant structural proper- ties is sequence-dependent bending and curvature. Numerous examples of sequence-induced DNA curva- ture have been described (e.g. [2]), all originating from functionally important regions of both prokaryotic and eukaryotic DNA. The majority of these loci play an important role in regulation of gene expression zyxwvu via DNA-protein interactions. Originally, DNA curvature was attributed to the occurrence of periodical d(A) tracts, which are in phase with the helical repeat of the DNA molecule [I, 31. It was later shown that a number of other sequence elements are also moderately curved, suggesting that sequence-induced curving in DNA may be a more general phenomenon [4, 51. In addition to fixed or static bending, sequence-dependent DNA flexi- bility is another inherent property which plays an impor- tant biological role and affects results of currently used DNA analysis techniques [6-91. Among methods to investigate DNA conformation, non- denaturing polyacrylamide gel electrophoresis has become widespread [4, 10-131. The anomalous mobility Correspondence: Professor Barry L. Karger, Barnett Institute, North- eastern University, Boston, MA 02115, USA (Tel: +617-373-2867; Fax: +617-373-2855) Abbreviations: EtBr, ethidium bromide; EthD, ethidium homodimer; LPA, linear polyacrylamide; TBE, Tris- boric acid - EDTA buffer; zyxwvut TO, thiazole orange Keywords: Capillary electrophoresis / DNA migration / DNA confor- mation of DNA fragments has generally been associated with DNA curvature or altered flexibility [14]. Since DNA mobility is directly related to the mean square end- to-end distance [15-171, curved DNA fragments will migrate more slowly than noncurved fragments [4]. Capillary electrophoresis has become an attractive method for the separation of DNA molecules due to its high resolution, speed, sensitivity, quantitation and the possibility of sample collection [18]. Capillary gel electro- phoresis, utilizing cross-linked polyacrylamide gel-filled columns, was first successfully used for separation of DNA molecules [ 191. Stability problems of gel-filled capillaries have been overcome by use of replaceable entangled polymer networks, such as linear polyacryla- mide (LPA) [20] and cellulose derivatives [21, 221. High resolution CE separations of double-stranded DNA fragments using polymer network matrices have been achieved [20, 23-25]. Our previous work demonstrated that the resolution of dsDNA fragments which differ as little as one base pair in length is possible in the range up to approximately 300 bp [20], but this work has also suggested cautious interpretation of experimental data in terms of resolution, where sequence-induced mobility changes might occur. These anomalous migration effects are enhanced with high electric field operation, as occurs in capillary electrophoresis. This paper explores the ano- malous migration behavior of several common dsDNA standards with replaceable polyacrylamide sieving matrices and describes the effect of various parameters on this behavior. The effects are general for sieving matrices, the extent of the anomalities dependent on the conditions employed. As an example of the generality, since completion of this work, a paper on anomalous DNA migration in capillary electrophoresis using a pro- prietary sieving matrix has appeared [26]. 2 Materials and methods 2.1 Apparatus The laboratory-built CE instrument was similar to that previously described [23]. A negative high voltage power supply (Model PO 434, Trek Inc., Medina, NY, USA) was * Present address: Hewlett Packard, Wilmington, DE. ** Presented in part at HPCE '94, San Diego, CA, February, 1994. 0 VCH Verlagsgesellschaft mbH, 69451 Weinheim, 1995 0173-0835/95/0303-0377 $5.00+.25/0