Improved methods for the formation and stabilization of R-loops.
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.
D001483:Base Sequence; D004247:DNA / Q000302:isolation & purification; D007700:Kinetics; D008854:Microscopy, Electron; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D011061:Poly A; D012441:Saccharomyces cerevisiae / Q000032:analysis; D014307:Trioxsalen
D011061:Poly A; D004247:DNA; D014307:Trioxsalen