Abstract
Two-dimensional (2D) gel electrophoresis employs distinct electrophoretic conditions to better resolve complex mixtures of molecules. In combination with Southern analysis, 2D agarose gel electrophoresis is routinely employed to detect and analyze DNA intermediates that arise during the replication and repair of chromosomes. By separating intermediates into their component single-strands, native/denaturing 2D gels can reveal structure that is not apparent under native conditions alone. Here, we describe a general method for native/denaturing two-dimensional gel electrophoresis and its application to understanding the DNA strand-composition of recombination intermediates formed during meiosis.
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References
Bell L, Byers B (1983) Separation of branched from linear DNA by two-dimensional gel electrophoresis. Anal Biochem 130:527
Bell LR, Byers B (1983) Homologous association of chromosomal DNA during yeast meiosis. Cold Spring Harb Symp Quant Biol 47(Pt 2):829–840
Brewer BJ, Fangman WL (1991) Mapping replication origins in yeast chromosomes. Bioessays 13:317
Dijkwel PA, Hamlin JL (1997) Mapping replication origins by neutral/neutral two-dimensional gel electrophoresis. Methods 13:235
Huberman JA (1997) Mapping replication origins, pause sites, and termini by neutral/alkaline two-dimensional gel electrophoresis. Methods 13:247
McDonell M, Simon MN, Studier FW (1977) Analysis of restriction fragments of T7 DNA and determination of molecular weights by electrophoresis in neutral and alkaline gels. J Mol Biol 110:119
Zakharyevich K et al (2010) Temporally and biochemically distinct activities of Exo1 during meiosis: double-strand break resection and resolution of double Holliday junctions. Mol Cell 40:1001
Wahls WP, DeWall KM, Davidson MK (2005) Mapping of ssDNA nicks within dsDNA genomes by two-dimensional gel electrophoresis. J Arkansas Acad Sci 59:178
Schwacha A, Kleckner N (1994) Identification of joint molecules that form frequently between homologs but rarely between sister chromatids during yeast meiosis. Cell 76:51
Lao JP, Oh SD, Shinohara M, Shinohara A, Hunter N (2008) Rad52 promotes post-invasion steps of meiotic double-strand-break repair. Mol Cell 29:517
Allers T, Lichten M (2001) Intermediates of yeast meiotic recombination contain heteroduplex DNA. Mol Cell 8:225
Schwacha A, Kleckner N (1995) Identification of double holliday junctions as intermediates in meiotic recombination. Cell 83:783
Cromie GA et al (2006) Single holliday junctions are intermediates of meiotic recombination. Cell 127:1167
Bzymek M, Thayer NH, Oh SD, Kleckner N, Hunter N (2010) Double holliday junctions are intermediates of DNA break repair. Nature 464:937
Oh SD et al (2007) BLM ortholog, Sgs1, prevents aberrant crossing-over by suppressing formation of multichromatid joint molecules. Cell 130:259
Oh SD, Lao JP, Taylor AF, Smith GR, Hunter N (2008) RecQ helicase, Sgs1, and XPF family endonuclease, Mus81-Mms4, resolve aberrant joint molecules during meiotic recombination. Mol Cell 31:324
Oh SD et al (2009) Stabilization and electrophoretic analysis of meiotic recombination intermediates in Saccharomyces cerevisiae. Methods Mol Biol 557:209
Green MR, Sambrook J (2012) Molecular cloning: a laboratory manual (fourth edition). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Ruven HJ, Seelen CM, Lohman PH, Mullenders LH, van Zeeland AA (1994) Efficient synthesis of 32P-labeled single-stranded DNA probes using linear PCR; application of the method for analysis of strand-specific DNA repair. Mutat Res 315:189
Acknowledgements
This work was supported by National Institutes of Health National Institute of General Medical Sciences (NIH NIGMS) grant GM074223 to N.H. N.H. is an Early Career Scientist of the Howard Hughes Medical Institute.
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Lao, J.P., Tang, S., Hunter, N. (2013). Native/Denaturing Two-Dimensional DNA Electrophoresis and Its Application to the Analysis of Recombination Intermediates. In: Makovets, S. (eds) DNA Electrophoresis. Methods in Molecular Biology, vol 1054. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-565-1_6
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DOI: https://doi.org/10.1007/978-1-62703-565-1_6
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