Two-dimensional strandness-dependent electrophoresis.
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 degrees 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.
Gunnarsson
Gudmundur H
GH
Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Iceland, IS-101 Reykjavik, Iceland.
Gudmundsson
Bjarki
B
Thormar
Hans G
HG
Alfredsson
Arni
A
Jonsson
Jon J
JJ
D020137:Base Pair Mismatch; D053904:DNA Breaks, Single-Stranded; D004249:DNA Damage; D004277:DNA, Single-Stranded / Q000302:isolation & purification*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D009692:Nucleic Acid Heteroduplexes / Q000302:isolation & purification*; D012107:Research Design
D004277:DNA, Single-Stranded; D009692:Nucleic Acid Heteroduplexes