Mapping site-specific endonuclease binding to DNA by direct imaging with AFM

PDF Version Also Available for Download.

Description

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 1,000 times greater than wild type enzyme but with cleavage rate constants reduced by a factor of 10{sup 4}, the authors demonstrate site-specific mapping by direct AFM imaging. Images are presented showing specific-site binding of Gln-111 to plasmids having either one (pBS{sup +}) or two (pMP{sup 32}) EcoRI sites. Identification of the Gln-111/DNA complex is greatly enhanced by biotinylation of the complex followed by reaction with streptavidin ... continued below

Physical Description

6 p.

Creation Information

Allison, D.P.; Thundat, T.; Doktycz, M.J.; Kerper, P.S.; Warmack, R.J.; Modrich, P. et al. December 31, 1995.

Context

This article is part of the collection entitled: Office of Scientific & Technical Information Technical Reports and was provided by UNT Libraries Government Documents Department to Digital Library, a digital repository hosted by the UNT Libraries. It has been viewed 11 times . More information about this article can be viewed below.

Who

People and organizations associated with either the creation of this article or its content.

Authors

Sponsor

Publisher

Provided By

UNT Libraries Government Documents Department

Serving as both a federal and a state depository library, the UNT Libraries Government Documents Department maintains millions of items in a variety of formats. The department is a member of the FDLP Content Partnerships Program and an Affiliated Archive of the National Archives.

Contact Us

What

Descriptive information to help identify this article. Follow the links below to find similar items on the Digital Library.

Description

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 1,000 times greater than wild type enzyme but with cleavage rate constants reduced by a factor of 10{sup 4}, the authors demonstrate site-specific mapping by direct AFM imaging. Images are presented showing specific-site binding of Gln-111 to plasmids having either one (pBS{sup +}) or two (pMP{sup 32}) 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 the 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.

Physical Description

6 p.

Notes

OSTI as DE96005989

Source

  • SPIE `95: SPIE conference on optics, electro-optics, and laser application in science, engineering and medicine, San Jose, CA (United States), 5-14 Feb 1995

Language

Item Type

Identifier

Unique identifying numbers for this article in the Digital Library or other systems.

  • Other: DE96005989
  • Report No.: CONF-950226--58
  • Grant Number: AC05-84OR21400
  • Office of Scientific & Technical Information Report Number: 201716
  • Archival Resource Key: ark:/67531/metadc672676

Collections

This article is part of the following collection of related materials.

Office of Scientific & Technical Information Technical Reports

What responsibilities do I have when using this article?

When

Dates and time periods associated with this article.

Creation Date

  • December 31, 1995

Added to The UNT Digital Library

  • June 29, 2015, 9:42 p.m.

Description Last Updated

  • Jan. 19, 2016, 7:41 p.m.

Usage Statistics

When was this article last used?

Yesterday: 0
Past 30 days: 0
Total Uses: 11

Interact With This Article

Here are some suggestions for what to do next.

Start Reading

PDF Version Also Available for Download.

Citations, Rights, Re-Use

Allison, D.P.; Thundat, T.; Doktycz, M.J.; Kerper, P.S.; Warmack, R.J.; Modrich, P. et al. Mapping site-specific endonuclease binding to DNA by direct imaging with AFM, article, December 31, 1995; Tennessee. (digital.library.unt.edu/ark:/67531/metadc672676/: accessed August 19, 2017), University of North Texas Libraries, Digital Library, digital.library.unt.edu; crediting UNT Libraries Government Documents Department.