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Two-dimensional strandness-dependent electrophoresis

Nature Protocols volume 1, pages 3011–3018 (2006)Cite this article

Abstract

Two-dimensional strandness-dependent electrophoresis (2D-SDE) separates nucleic acids in complex samples according to strandness, conformation and length. Under the non-denaturing conditions of the first electrophoretic step, single-stranded DNA, double-stranded DNA and RNA·DNA hybrids of similar length migrate at different rates. The second electrophoretic step is performed under denaturing conditions (7 mol l−1 urea, 55 °C) so that all the molecules are single-stranded and separate according to length only. 2D-SDE is useful for revealing important characteristics of complex nucleic acid samples in manipulations such as amplification, renaturation, cDNA synthesis and microarray hybridization. It can also be used to identify mispaired, nicked or damaged fragments in double-stranded DNA. The protocol takes approximately 2 h and requires only basic skills, equipment and reagents.

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Figure 1: The principle of 2D-SDE.
Figure 2: Separation of nick-containing DNA from intact dsDNA using nick-dependent electrophoresis.
Figure 3: Comparison of agarose and 2D-SDE analysis of highly complex genome representations generated with selective PCR amplification of short restriction fragments from human genomic DNA.
Figure 4: 2D-SDE analysis of four different complex nucleic acid samples.
Figure 5: Picture of a typical setup for horizontal electrophoresis in the second dimension using a Multiphor apparatus.

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Acknowledgements

This work was supported by the Icelandic Research Council, the University of Iceland Research Fund, the Science Fund of Landspitali-University Hospital and BioCule Inc. BioCule Inc. has applied for a patent on the method. Authors G.H.G., B.G., H.G.T. and J.J.J. own stock and B.G. and H.G.T. are currently employed by BioCule. BioCule also funds research projects in J.J.J.'s research laboratory.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Iceland, Reykjavik, IS-101, Iceland

    Gudmundur H Gunnarsson, Hans G Thormar, Arni Alfredsson & Jon J Jonsson

  2. BioCule Inc., Reykjavik, IS-101, Iceland

    Gudmundur H Gunnarsson, Bjarki Gudmundsson & Hans G Thormar

  3. Department of Genetics and Molecular Medicine, Landspitali-University Hospital, Reykjavik, IS-101, Iceland

    Jon J Jonsson

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Correspondence to Jon J Jonsson.

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BioCule Inc. has applied for a patent on the method. Authors GHG, BG, HGT and JJJ own stock and BG and HGT are currently employed by BioCule. BioCule also funds research projects in JJJ's research laboratory.

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Gunnarsson, G., Gudmundsson, B., Thormar, H. et al. Two-dimensional strandness-dependent electrophoresis. Nat Protoc 1, 3011–3018 (2006). https://doi.org/10.1038/nprot.2006.477

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