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. 1979 Jun 11;6(7):2499–2517. doi: 10.1093/nar/6.7.2499

Improved methods for the formation and stabilization of R-loops

David B Kaback 1, Lynne M Angerer 1, Norman Davidson 1
PMCID: PMC327868  PMID: 379821

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.

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