HIGH-RESOLUTION MAPPING DNAS BY R-LOOP ATOMIC-FORCE MICROSCOPY

R-loops formed by short RNA transcripts have been imaged by atomic for ce microscopy (AFM) at a constant force in the height mode. The techni que was applied to mapping the human endogenous retrovirus K10 family (HERV-K10) long terminal repeats (LTR) within individual plasmids and cosmids. RNA probes specific for the U3 (384 nt) and U5 (375 nt) LTR r egions separated by a span of 200 bp were used for R-loop formation wi th LTRs located within plasmid (3.8 kb) or cosmid (similar to 40 kb) D NAs. R-loops stabilized by:glyoxal treatment and adsorbed onto the mic a surface in the presence of magnesium ions looked like looped out seg ments of RNA:DNA hybrids. The total yield of R-loops was usually simil ar to 95%. The RNA:DNA hybrids were found to be 12-15% shorter than th e corresponding DNA:DNA duplex. The two regions of the LTR could be ea sily discerned in the AFM images as clearly separated loops. R-loop po sitions determined on cosmids by AFM were accurate to similar to 0.5% of the cosmid length. This technique might be easily adapted for mappi ng various sequences such as gene exons or regulatory regions and for detecting insertions, deletions and rearrangements that cause human ge netic diseases.