The analysis of nucleic acid structure by zone electrophoresis in agarose gels has become an increasingly important method in studies requiring the separation and identification of species differing only slightly in molecular weight or conformation. We have demonstrated the existence of several circular DNA forms that appear in cells infected with the bacteriophage ΦX174.
Wt/am3 heteroduplex molecules containing a single base mismatch (relaxed RFI molecules) were constructed in vitro, purified by sedimentation, and analyzed by zone electrophoresis on 1.4% agarose gels. In order to verify that the band of denaturation-resistant heteroduplex was indeed relaxed RFI, we compared it with relaxed RFI prepared from supercoiled RFI using the DNA relaxing enzyme (DNA topoisomerase) from Agrobacterium tumefaciens. To distinguish the product of the reaction from RF II molecules, it was analyzed by electrophoresis in 1.4% agarose with 0.5 μg/ml ethidium bromide. Under these conditions supercoiled RFI, relaxed RFI, RFII, RFIII and single-stranded DNA all migrate at different rates and can be readily identified with appropriate markers. Multiple length rings were also observed.
Much of the analyses of the mobilities confirmed our understanding of DNA migration in electrophoresis. The relaxed RFI migrates more rapidly than natural supercoiled REI in gels containing ethidium. In the absence of ethidium the supercoiled molecules migrate faster than relaxed RFI. Contrary to expectations, in the presence of ethidium bromide, the relaxed RFI and supercoiled RFI exchange positions.
Anthony J. Zuccarelli
Robert L. Nutter
Robert W. Teel
Master of Arts (MA)
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This title appears here courtesy of the author, who has granted Loma Linda University a limited, non-exclusive right to make this publication available to the public. The author retains all other copyrights.
Hanson, Virginia McKee, "Electrophoretic Mobilities of Topoisomers of ΦX174 DNA" (1981). Loma Linda University Electronic Theses, Dissertations & Projects. 1384.
Loma Linda University Electronic Theses and Dissertations
Loma Linda University. Del E. Webb Memorial Library. University Archives