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The electronic form of this document may be cited in the following style:
Human Genome Program, U.S. Department of Energy, DOE Human Genome Program Contractor-Grantee Workshop IV, 1994.

Abstracts scanned from text submitted for November 1994 DOE Human Genome Program Contractor-Grantee Workshop. Inaccuracies have not been corrected.

INTERACTION OF DIMERIC INTERCALATING FLUORESCENT DYES WITH SINGLE-STRANDED DNA

Hays S. Rye and Alexander N. Glazer,
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720.

We have shown that a wide variety of suitably designed dimeric dyes form highly fluorescent complexes with double-stranded DNA (dSDNA) stable to electrophoresis[1], and that such dsDNA-dye complexes can be exploited for the high-sensitivity determination of dsDNA concentration in solution[2], for multiplex detection of restriction fragments in slab gel and capillary electrophoresis[1,3,5], and in the quantitative study of protein-DNA interactions[6]. Others have demonstrated that these dimeric dyes can be utilized for the ultra-high sensitivity analysis of dsDNA content in cells by flow cytometry[7], in the analysis of PCR products[8], and in the examination of the static and dynamic properties of isolated DNA molecules[9,10].

To extend the range of applications of such dye-DNA complexes, we have initiated studies of the interaction with single-stranded DNA (ssDNA) of dimeric dyes capable of bisintercalation, such as ethidium homodimer (EthD) and thiazole orange homodimer (TOTO), that form stable complexes with dsDNA[3,11]. These studies compared in detail the binding of these dyes to linearized M13 ss and dsDNAs and characterized both types of complexes by absorption, fluorescence, and circular dichroism spectroscopy. Surprisingly, M13 ss and dsDNAs bind the dyes with similar affinity at comparable numbers of high affinity sites. Per bound dye, the fluorescence emission of the ssDNA-dye complexes is about 3-fold weaker than that of the corresponding dsDNA complexes. Both TOTO and EthD form complexes with M13 ssDNA stable to electrophoresis. The ssDNA-dye complexes are more stable at high Na+ concentrations than the corresponding dsDNA complexes. Such ssDNA-dye complexes, preformed at 1 dye per 15 bases, retain about 50% of the bound dye when challenged with a 600-fold by weight excess of unlabeled dsDNA. These preliminary results indicate that the applications of the fluorescent DNA dye complexes can be extended to ssDNA-complexes.

Supported by a grant from the Director, Office of Energy Research, Office of Health and Environmental Research of the U.S. Department of Energy under contract DE-FG-91-61125.

[1]A.N. Glazer and H.S. Rye, Nature (London) 359, 859-861 (1992).
[2]H. S. Rye, J.M. Dabora, M.A. Quesada, R. A. Mathies, and A.N. Glazer, Anal. Biochem. 207, 144-150 (1993).
[3]H.S. Rye, S. Yue, D.E. Wemmer, M.A. Quesada, R.P. Haugland, R.A. Mathies, and A.N. Glazer, Nucleic Acids Res. 20, 3803-2812 (1992).
[4]S.C. Benson, R.A. Mathies, and A.N. Glazer, Nucleic Acids Res. 21, 5720-5726 (1993).
[5]H. Zhu, S.M. Clark, S.C. Benson, H.S. Rye, A.N. Glazer, and R.A. Mathies, Anal Chem. 66, 1941-1948 (1994).
[6]H.S. Rye, B.L. Drees, H.C.M. Nelson, and A.N. Glazer, J. Biol. Chem. 268, 25229-25238 (1993).
[7]G.T. Hirons, J.J. Fawcett, and H.A. Crissman, Cytometry 15, 129-140 (1994).
[8]K. Srinivasan, S.C. Morris, J.E. Girard, M.C. Kline, and D.J. Reeder, Appl. Theoret. Electrophor. 3, 235-239 (1993).
[9]I. Auzanneau, C. Barreau, and L. Salome, Compt. Rend. Acad. Sci.., Ser III 316, 459-462 (1993)
[10]T.T. Perkins, D.E. Smith, and S. Chu, Science 264, 822-826 (1994).
[11]A.N. Glazer, K. Peck and R.A. Mathies, Proc. Natl. Acad. Sci. USA 87, 3851-3855 (1990).