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

D. P. Allison; P. S. Kerper; M. J. Doktycz; J. A. Spain; P. Modrich; F. W. Larimer; T. Thundat; R. J. Warmack

doi:10.1073/pnas.93.17.8826

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

D. P. Allison, P. S. Kerper, M. J. Doktycz, J. A. Spain, P. Modrich, F. W. Larimer, T. Thundat, R. J. Warmack

Sensors and Electronics

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Abstract

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 10⁴. ScaI-linearized plasmids with single (pBS⁺) and double (pGEM-luc and pSV-β-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.

Original language	English
Pages (from-to)	8826-8829
Number of pages	4
Journal	Proceedings of the National Academy of Sciences of the United States of America
Volume	93
Issue number	17
DOIs	https://doi.org/10.1073/pnas.93.17.8826
State	Published - Aug 20 1996

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Allison, D. P., Kerper, P. S., Doktycz, M. J., Spain, J. A., Modrich, P., Larimer, F. W., Thundat, T., & Warmack, R. J. (1996). Direct atomic force microscope imaging of EcoRI endonuclease site specifically bound to plasmid DNA molecules. Proceedings of the National Academy of Sciences of the United States of America, 93(17), 8826-8829. https://doi.org/10.1073/pnas.93.17.8826

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title = "Direct atomic force microscope imaging of EcoRI endonuclease site specifically bound to plasmid DNA molecules",

abstract = "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 104. ScaI-linearized plasmids with single (pBS+) and double (pGEM-luc and pSV-β-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.",

author = "Allison, {D. P.} and Kerper, {P. S.} and Doktycz, {M. J.} and Spain, {J. A.} and P. Modrich and Larimer, {F. W.} and T. Thundat and Warmack, {R. J.}",

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Allison, DP, Kerper, PS, Doktycz, MJ, Spain, JA, Modrich, P, Larimer, FW, Thundat, T & Warmack, RJ 1996, 'Direct atomic force microscope imaging of EcoRI endonuclease site specifically bound to plasmid DNA molecules', Proceedings of the National Academy of Sciences of the United States of America, vol. 93, no. 17, pp. 8826-8829. https://doi.org/10.1073/pnas.93.17.8826

TY - JOUR

T1 - Direct atomic force microscope imaging of EcoRI endonuclease site specifically bound to plasmid DNA molecules

AU - Allison, D. P.

AU - Kerper, P. S.

AU - Doktycz, M. J.

AU - Spain, J. A.

AU - Modrich, P.

AU - Larimer, F. W.

AU - Thundat, T.

AU - Warmack, R. J.

PY - 1996/8/20

Y1 - 1996/8/20

N2 - 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 104. ScaI-linearized plasmids with single (pBS+) and double (pGEM-luc and pSV-β-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.

AB - 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 104. ScaI-linearized plasmids with single (pBS+) and double (pGEM-luc and pSV-β-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.

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DO - 10.1073/pnas.93.17.8826

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VL - 93

SP - 8826

EP - 8829

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

IS - 17

ER -

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

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