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.
| Original language | English |
|---|---|
| Pages (from-to) | 3011-3018 |
| Number of pages | 8 |
| Journal | Nature Protocols |
| Volume | 1 |
| Issue number | 6 |
| DOIs | |
| Publication status | Published - Jan 2007 |
Bibliographical note
Funding Information:ACKNOWLEDGMENTS 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.