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Restriction enzyme kinetics monitored by UV linear dichroism
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Hicks, Matthew R., Rodger, Alison, Thomas, Christopher M., Batt, Sarah M. and Dafforn, Tim (2006) Restriction enzyme kinetics monitored by UV linear dichroism. Biochemistry, Volume 45 (Number 29). pp. 8912-8917. doi:10.1021/bi0601712 ISSN 0006-2960.
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Official URL: http://dx.doi.org/10.1021/bi0601712
Abstract
The use of linear dichroism (LD) spectroscopy for biological applications has been brought to the forefront recently by our development of thermostated microvolume Couette cells. We present a method for following the digestion of DNA by restriction endonucleases in real time without the use of any extrinsic dyes or labels. This is accomplished using linear dichroism spectroscopy (the differential absorbance of light polarized parallel and perpendicular to the sample orientation axis). The differential absorbance signal depends on the degree of alignment of the molecules. In this case the DNA is aligned by Couette flow (flowing the solution in the annular gap between two concentric cylinders), and we monitor the increase in alignment upon linearization of a circular DNA molecule. In addition, we observe a decrease in alignment upon further digestion and subsequent shortening of the DNA. Ten enzymes were investigated: seven enzymes with a single cut site (EcoRI, KpnI, NdeI, NotI, NruI, SmaI, XbaI), two enzymes with two cut sites (BstZ17I, EagI), and one enzyme with no cut site (ClaI). LD, as implemented in this new assay, is broadly applicable across a wide range of DNA-modifying enzymes and compounds and, as such, is a useful addition to the toolbox of biological characterization.
Item Type: | Journal Article | ||||
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Subjects: | Q Science > QD Chemistry | ||||
Divisions: | Faculty of Science, Engineering and Medicine > Science > Chemistry | ||||
Journal or Publication Title: | Biochemistry | ||||
Publisher: | American Chemical Society | ||||
ISSN: | 0006-2960 | ||||
Official Date: | 25 July 2006 | ||||
Dates: |
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Volume: | Volume 45 | ||||
Number: | Number 29 | ||||
Number of Pages: | 6 | ||||
Page Range: | pp. 8912-8917 | ||||
DOI: | 10.1021/bi0601712 | ||||
Status: | Peer Reviewed | ||||
Publication Status: | Published | ||||
Access rights to Published version: | Restricted or Subscription Access |
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