Open AccessDirect atomic force microscope imaging of EcoRI endonuclease site specifically bound to plasmid DNA molecules.
Author(s) -
David P. Allison,
Peggy S. Kerper,
Mitchel J. Doktycz,
Jaume Spain,
Paul Modrich,
Frank W. Larimer,
Thomas Thundat,
R. J. Warmack
Publication year - 1996
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.93.17.8826
Subject(s) - ecori , restriction enzyme , plasmid , endonuclease , dna , chemistry , microbiology and biotechnology , mutant , cleavage (geology) , binding site , recombinant dna , microscope , biology , biophysics , biochemistry , gene , physics , optics , paleontology , fracture (geology)
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.