Mapping site-specific endonuclease binding to DNA by direct imaging with atomic force microscopy (AFM)

David P. Allison; Thomas G. Thundat; P. Modrich; R. J. Isfort; Mitchel J. Doktycz; P. S. Kerper; R. J. Warmack

doi:10.1117/12.206037

Mapping site-specific endonuclease binding to DNA by direct imaging with atomic force microscopy (AFM)

David P. Allison, Thomas G. Thundat, P. Modrich, R. J. Isfort, Mitchel J. Doktycz, P. S. Kerper, R. J. Warmack

Sensors and Electronics

Research output: Chapter in Book/Report/Conference proceeding › Conference contribution › peer-review

1 Scopus citations

Abstract

Physical mapping of DNA can be accomplished by direct AFM imaging of site specific proteins bound to DNA molecules. Using Gln-111, a mutant of EcoRI endonuclease with a specific affinity for EcoRI sites 1000 times greater than wild type enzyme but with cleavage rate constants reduced by a factor of 10⁴, we demonstrate site-specific mapping by direct AFM imaging. Images are presented showing specific-site binding of Gln-111 to plasmids having either one (pBS⁺) or two (pMP³²) EcoRI sites. Identification of the Gln-111/DNA complex is greatly enhanced by biotinylation of the complex followed by reaction with streptavidin gold prior to imaging. Image enhancement coupled with improvements in our preparation techniques for imaging large DNA molecules, such as lambda DNA (47 kb), has the potential to contribute to direct AFM restriction mapping of cosmid-sized genomic DNAs.

Original language	English
Title of host publication	Proceedings of SPIE - The International Society for Optical Engineering
Publisher	Society of Photo-Optical Instrumentation Engineers
Pages	24-29
Number of pages	6
Volume	2386
ISBN (Print)	0819417335, 9780819417336
DOIs	https://doi.org/10.1117/12.206037
State	Published - 1995
Event	Ultrasensitive Instrumentation for DNA Sequencing and Biochemical Diagnostics - San Jose, CA, USA Duration: Feb 8 1995 → Feb 10 1995

Conference

Conference	Ultrasensitive Instrumentation for DNA Sequencing and Biochemical Diagnostics
City	San Jose, CA, USA
Period	02/8/95 → 02/10/95

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10.1117/12.206037

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Allison, D. P., Thundat, T. G., Modrich, P., Isfort, R. J., Doktycz, M. J., Kerper, P. S., & Warmack, R. J. (1995). Mapping site-specific endonuclease binding to DNA by direct imaging with atomic force microscopy (AFM). In Proceedings of SPIE - The International Society for Optical Engineering (Vol. 2386, pp. 24-29). Society of Photo-Optical Instrumentation Engineers. https://doi.org/10.1117/12.206037

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title = "Mapping site-specific endonuclease binding to DNA by direct imaging with atomic force microscopy (AFM)",

abstract = "Physical mapping of DNA can be accomplished by direct AFM imaging of site specific proteins bound to DNA molecules. Using Gln-111, a mutant of EcoRI endonuclease with a specific affinity for EcoRI sites 1000 times greater than wild type enzyme but with cleavage rate constants reduced by a factor of 104, we demonstrate site-specific mapping by direct AFM imaging. Images are presented showing specific-site binding of Gln-111 to plasmids having either one (pBS+) or two (pMP32) EcoRI sites. Identification of the Gln-111/DNA complex is greatly enhanced by biotinylation of the complex followed by reaction with streptavidin gold prior to imaging. Image enhancement coupled with improvements in our preparation techniques for imaging large DNA molecules, such as lambda DNA (47 kb), has the potential to contribute to direct AFM restriction mapping of cosmid-sized genomic DNAs.",

author = "Allison, {David P.} and Thundat, {Thomas G.} and P. Modrich and Isfort, {R. J.} and Doktycz, {Mitchel J.} and Kerper, {P. S.} and Warmack, {R. J.}",

year = "1995",

doi = "10.1117/12.206037",

language = "English",

isbn = "0819417335",

volume = "2386",

pages = "24--29",

booktitle = "Proceedings of SPIE - The International Society for Optical Engineering",

publisher = "Society of Photo-Optical Instrumentation Engineers",

note = "Ultrasensitive Instrumentation for DNA Sequencing and Biochemical Diagnostics ; Conference date: 08-02-1995 Through 10-02-1995",

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Allison, DP, Thundat, TG, Modrich, P, Isfort, RJ, Doktycz, MJ, Kerper, PS & Warmack, RJ 1995, Mapping site-specific endonuclease binding to DNA by direct imaging with atomic force microscopy (AFM). in Proceedings of SPIE - The International Society for Optical Engineering. vol. 2386, Society of Photo-Optical Instrumentation Engineers, pp. 24-29, Ultrasensitive Instrumentation for DNA Sequencing and Biochemical Diagnostics, San Jose, CA, USA, 02/8/95. https://doi.org/10.1117/12.206037

Mapping site-specific endonuclease binding to DNA by direct imaging with atomic force microscopy (AFM). / Allison, David P.; Thundat, Thomas G.; Modrich, P. et al.
Proceedings of SPIE - The International Society for Optical Engineering. Vol. 2386 Society of Photo-Optical Instrumentation Engineers, 1995. p. 24-29.

Research output: Chapter in Book/Report/Conference proceeding › Conference contribution › peer-review

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AU - Allison, David P.

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AU - Doktycz, Mitchel J.

AU - Kerper, P. S.

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AB - Physical mapping of DNA can be accomplished by direct AFM imaging of site specific proteins bound to DNA molecules. Using Gln-111, a mutant of EcoRI endonuclease with a specific affinity for EcoRI sites 1000 times greater than wild type enzyme but with cleavage rate constants reduced by a factor of 104, we demonstrate site-specific mapping by direct AFM imaging. Images are presented showing specific-site binding of Gln-111 to plasmids having either one (pBS+) or two (pMP32) EcoRI sites. Identification of the Gln-111/DNA complex is greatly enhanced by biotinylation of the complex followed by reaction with streptavidin gold prior to imaging. Image enhancement coupled with improvements in our preparation techniques for imaging large DNA molecules, such as lambda DNA (47 kb), has the potential to contribute to direct AFM restriction mapping of cosmid-sized genomic DNAs.

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ER -

Mapping site-specific endonuclease binding to DNA by direct imaging with atomic force microscopy (AFM)

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