An Easy Method to Non-Destructively Separate the Strands of a Circular Duplex Plasmid Chromosome: A Preliminary Report
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Abstract
A straightforward and cost-effective method has been developed for the non-destructive separation of the two strands of a circular duplex plasmid chromosome. This method was adapted from the original protocol reported by Wu and Wu (1996), who achieved strand separation using prolonged low-current electrophoresis. In contrast, the method described here utilizes a two-step denaturation process: initial alkali denaturation to pH 13 to generate “Form IV” DNA, followed by denaturation in 90% formamide at 80°C under low-salt conditions. The resulting single strands were observed to migrate as two discrete bands on agarose gel electrophoresis, indicating successful strand separation without degradation. This approach offers a reproducible, rapid, and inexpensive alternative for investigating the topology and helicity of circular DNA molecules.
Keywords
DNA topology, plasmid, Form IV, strand separation, non-helical DNA, circular DNA
Article Details
Supporting Agencies
This report is dedicated to my late wife, Anita Shatz, who loved me during her life, and who funded this study in her death.
How to Cite
Biegeleisen, K. (2025). An Easy Method to Non-Destructively Separate the Strands of a Circular Duplex Plasmid Chromosome: A Preliminary Report. Chemical Reports, 5(1), 298-307. https://doi.org/10.25082/CR.2024.01.005
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
References
- Stettler UH, Weber H, Koller Th, et al. Preparation and characterization of form V DNA, the duplex DNA resulting from association of complementary, circular single-stranded DNA. Journal of Molecular Biology. 1979, 131(1): 21-40. https://doi.org/10.1016/0022-2836(79)90299-7
- Crick FHC, Wang JC, Bauer WR. Is DNA really a double helix? Journal of Molecular Biology. 1979, 129(3): 449-461. https://doi.org/10.1016/0022-2836(79)90506-0
- Wu R, Wu TT. A novel intact circular dsDNA supercoil. Bulletin of Mathematical Biology. 1996, 58(6): 1171-1185. https://doi.org/10.1007/bf02458388
- Xu YC. Finding of a zero linking number topoisomer. Biochimica et Biophysica Acta (BBA) - General Subjects. 2009, 1790(2): 126-133. https://doi.org/10.1016/j.bbagen.2008.10.012
- Biegeleisen K. The probable structure of the protamine–DNA complex. Journal of Theoretical Biology. 2006, 241(3): 533-540. https://doi.org/10.1016/j.jtbi.2005.12.015
- Vinograd J, Lebowitz J, Radloff R, et al. The twisted circular form of polyoma viral DNA. Proceedings of the National Academy of Sciences. 1965, 53(5): 1104-1111. https://doi.org/10.1073/pnas.53.5.1104
- Strider W, Warner RC. Denatured Replicative form and Complex DNA OF φX174-Isolation and Renaturation//Federation Proceedings. 9650 Rockville Pike, Bethesda, MD 20814-3998: Federation Amer Soc Exp Biol. 1971, 30(3): 1053.
- Strider W. Denatured replicative form and complex DNA of φx174: isolation, renaturation, and sedimentation properties. Dissertation, New York University School of Medicine. Available by interlibrary loan. 1971.
- Strider W, Camien MN, Warner RC. Renaturation of denatured, covalently closed circular DNA. Journal of Biological Chemistry. 1981, 256(15): 7820-7829. https://doi.org/10.1016/s0021-9258(18)43352-2
- Rush MG, Warner RC. Alkali Denaturation of Covalently Closed Circular Duplex Deoxyribonucleic Acid. Journal of Biological Chemistry. 1970, 245(10): 2704-2708. https://doi.org/10.1016/s0021-9258(18)63125-4
- Biegeleisen K. The Probable Structure of “Form IV” (Alkali-Denatured Circular DNA). OALib. 2016, 03(10): 1-24. https://doi.org/10.4236/oalib.1103114
- Rodley GA, Scobie RS, Bates RH, et al. A possible conformation for double-stranded polynucleotides. Proceedings of the National Academy of Sciences. 1976, 73(9): 2959-2963. https://doi.org/10.1073/pnas.73.9.2959
- Sasisekharan V, Pattabiraman N. Double stranded polynucleotides: two typical alternative conformations for nucleic acids. Current Science. 1976, 45: 779-783. https://repository.ias.ac.in/49578/1/49578.pdf
- Wu TT. Secondary Structures of DNA. Proceedings of the National Academy of Sciences. 1969, 63(2): 400-405. https://doi.org/10.1073/pnas.63.2.400
- Cyriax B, Gäth R. The conformation of double-stranded DNA. Naturwissenschaften. 1978, 65(2): 106-108. https://doi.org/10.1007/bf00440551
- Delmonte C, Mann L. Variety in DNA secondary structure. Current Science. 2003, 85:1564-1570. https://www.currentscience.ac.in/Volumes/85/11/1564.pdf
- Pauling L, Corey RB. A Proposed Structure For The Nucleic Acids. Proceedings of the National Academy of Sciences. 1953, 39(2): 84-97. https://doi.org/10.1073/pnas.39.2.84
- Pouwels PH, Knijnenburg CM, van Rotterdam J, et al. Structure of the replicative form of bacteriophage φX174. Journal of Molecular Biology. 1968, 32(2): 169-182. https://doi.org/10.1016/0022-2836(68)90002-8
