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Published online 3 February 2004

Nucleic Acids Research, 2004, Vol. 32, No. 2 e23
© 2004 Oxford University Press

Two-dimensional conformation-dependent electrophoresis (2D-CDE) to separate DNA fragments containing unmatched bulge from complex DNA samples

Gudmundur H. Gunnarsson1,2, Hans G. Thormar1,3, Bjarki Gudmundsson2, Lina Akesson1 and Jon J. Jonsson*,1,3

1 Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Iceland, IS-101 Reykjavik, Iceland, 2 BioCule, IS-101 Reykjavik, Iceland and 3 Department of Genetics and Molecular Medicine, Landspitali-University Hospital, IS-101 Reykjavik, Iceland

*To whom correspondence should be addressed at Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Iceland, IS-101 Reykjavik, Iceland. Tel: +354 698 5995; Fax: +354 525 4886; Email: jonjj{at}hi.is

DNA fragments containing mispaired and modified bases, bulges, lesions and specific sequences have altered conformation. Methods for separating complex samples of DNA fragments based on conformation but independent of length have many applications, including (i) separation of mismatched or unmatched DNA fragments from those perfectly matched; (ii) simultaneous, diagnostic, mismatch scanning of multiple fragments; (iii) isolation of damaged DNA fragments from undamaged fragments; and (iv) estimation of reannealing efficiency of complex DNA samples. We developed a two-dimensional conformation-dependent electrophoresis (2D-CDE) method for separating DNA fragments based on length and conformation in the first dimension and only on length in the second dimension. Differences in migration velocity due to conformation were minimized during second dimension electrophoresis by introducing an intercalator. To test the method, we constructed 298 bp DNA fragments containing cytosine bulges ranging from 1 to 5 nt. Bulge-containing DNA fragments had reduced migration velocity in the first dimension due to altered conformation. After 2D-CDE, bulge-containing DNA fragments had migrated in front of an arc comprising heterogeneous fragments with regular conformation. This simple and robust method could be used in both analytical and preparative applications involving complex DNA samples.


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