Published January 11, 1979
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Improved methods for the formation and stabilization of R-loops
Abstract
Improved methods for the formation and stabilization of R-loops for visualization in the electron microscope are presented. The two complementary strands of a duplex DNA are photochemically crosslinked once every 1 to 3 kb using 4, 5', 8 trimethylpsoralen. R-loops are then formed by incubation with RNA in 70% formamide at a temperature above the DNA melting temperature. Finally, the R-loops are stabilized by modifying the free single strand of DNA with glyoxal, thus minimizing the displacement of the hybridized RNA by branch migration. In this manner R-loops can be formed and visualized at a high frequency irrespective of the base composition of the nucleic acid of interest.
Additional Information
Copyright © 1979 Oxford University Press. Received 23 January 1979. We are indebted to Fiona Gibson, Lynna Hereford and Michael Rosbash for Poly(A)+ RNA; the Paul D. Elder Co. for Trioxsalen; and Peter Chandler, James Casey and Pauline Yen for valuable discussions. DBK was supported by a Damon Runyon-Walter Winchell Cancer Research Foundation Fellowship and an U.S.P.H.S. National Research Service Award. This research was supported by grants GM 10991 and GM 20927 from the U.S.P.H.S. and RR 07003, Biomedical Research Grant.Files
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