DIRECT ATOMIC-FORCE MICROSCOPE IMAGING OF ECORI ENDONUCLEASE SITE-SPECIFICALLY BOUND TO PLASMID DNA-MOLECULES

Direct imaging with the atomic force microscope has been used to ident ify specific nucleotide sequences in plasmid DNA molecules, This was a ccomplished using EcoRI(Gln-lll), a mutant of the restriction enzyme t hat has a 1000-foId greater binding affinity than the wild-type enzyme but with cleavage rate constants reduced by a Factor of 10(4). ScaI-l inearized plasmids with single (pBS(+)) and double (pGEM-luc and pSV-b eta-galactosidase) EcoRI recognition sites were imaged, and the bound enzyme,vas localized to a 50- to 100-nt resolution. The high affinity for the EcoRI binding site exhibited by this mutant endonuclease, coup led with an observed low level of nonspecific binding, should prove va luable for physically mapping large DNA clones by direct atomic force microscope imaging.