Nucleic Acids Research, Vol 26, Issue 20 4603-4610, Copyright © 1998 by Oxford University Press
DV Klinov, IV Lagutina, VV Prokhorov, T Neretina, PP Khil, YB Lebedev, DI Cherny, VV Demin and ED Sverdlov
R-loops formed by short RNA transcripts have been imaged by atomic force
microscopy (AFM) at a constant force in the height mode. The technique 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 regions separated
by a span of 200 bp were used for R-loop formation with LTRs located within
plasmid (3.8 kb) or cosmid ( approximately 40 kb) DNAs. R-loops stabilized
by glyoxal treatment and adsorbed onto the mica surface in the presence of
magnesium ions looked like looped out segments of RNA:DNA hybrids. The
total yield of R-loops was usually approximately 95%. The RNA:DNA hybrids
were found to be 12- 15% shorter than the corresponding DNA:DNA duplex. The
two regions of the LTR could be easily discerned in the AFM images as
clearly separated loops. R-loop positions determined on cosmids by AFM were
accurate to approximately 0.5% of the cosmid length. This technique might
be easily adapted for mapping various sequences such as gene exons or
regulatory regions and for detecting insertions, deletions and
rearrangements that cause human genetic diseases.
ARTICLES
High resolution mapping DNAs by R-loop atomic force microscopy
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry and Institute of Molecular Genetics,Russian Academy of Sciences, Miklukho-Maklaya 16/10, Moscow 117871, Russia.
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