RESUMO
Direct imaging with the atomic force microscope has been used to identify specific nucleotide sequences in plasmid DNA molecules. This was accomplished using EcoRI (Gln-111), a mutant of the restriction enzyme that has a 1000-fold greater binding affinity than the wild-type enzyme but with cleavage rate constants reduced by a factor of 10(4). ScaI-linearized plasmids with single (pBS+) and double (pGEM-luc and pSV-beta-galactosidase) EcoRI recognition sites were imaged, and the bound enzyme was localized to a 50- to 100-nt resolution. The high affinity for the EcoRI binding site exhibited by this mutant endonuclease, coupled with an observed low level of nonspecific binding, should prove valuable for physically mapping large DNA clones by direct atomic force microscope imaging.
Assuntos
Mapeamento Cromossômico/métodos , Desoxirribonuclease EcoRI/ultraestrutura , Microscopia de Força Atômica/métodos , Plasmídeos/ultraestrutura , Análise de Sequência/métodos , Sítios de Ligação , Desoxirribonuclease EcoRI/genética , MutaçãoRESUMO
We present a technique, "snapshot blotting," for the electrophoretic transfer of nucleic acids and nucleoprotein complexes in gel electrophoresis bands onto highly stable carbon film-coated grids for imaging by electron microscopy. The method permits structural analysis of macromolecular species that have been resolved by a gel mobility-shift assay. To demonstrate the efficiency and integrity of the transfer process for a multiprotein-DNA assembly, we have imaged various species of a prokaryotic transcription complex, using the cleavage-defective EcoRI(Q111) protein as an orientation marker and as a blockade of transcription elongation. Snapshot blotting should be of great utility in the structural characterization of nucleic acids and protein-nucleic acid interactions.