Direct atomic force microscope imaging of EcoRI endonuclease site specifically bound to plasmid DNA molecules.

Published

Journal Article

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.

Full Text

Duke Authors

Cited Authors

  • Allison, DP; Kerper, PS; Doktycz, MJ; Spain, JA; Modrich, P; Larimer, FW; Thundat, T; Warmack, RJ

Published Date

  • August 20, 1996

Published In

Volume / Issue

  • 93 / 17

Start / End Page

  • 8826 - 8829

PubMed ID

  • 8799111

Pubmed Central ID

  • 8799111

International Standard Serial Number (ISSN)

  • 0027-8424

Digital Object Identifier (DOI)

  • 10.1073/pnas.93.17.8826

Language

  • eng

Conference Location

  • United States