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Direct observation of enzymes replicating DNA using a single-molecule DNA stretching assay

Journal Article


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Abstract


  • We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. Linearized λ DNA is modified to have a biotin on the end of one strand, and a digoxigenin moiety on the other end of the same strand. The biotinylated end is attached to a functionalized glass coverslip and the digoxigeninated end to a small bead. The assembly of these DNA-bead tethers on the surface of a flow cell allows a laminar flow to be applied to exert a drag force on the bead. As a result, the DNA is stretched close to and parallel to the surface of the coverslip at a force that is determined by the flow rate (Figure 1). The length of the DNA is measured by monitoring the position of the bead. Length differences between single- and double-stranded DNA are utilized to obtain real-time information on the activity of the replication proteins at the fork. Measuring the position of the bead allows precise determination of the rates and processivities of DNA unwinding and polymerization (Figure 2). © JoVE 2006-2011 All Rights Reserved.

Authors


  •   Kulczyk, Arkadiusz W. (external author)
  •   Tanner, Nathan A. (external author)
  •   Loparo, Joseph J. (external author)
  •   Richardson, Charles C. (external author)
  •   van Oijen, Antoine M.

Publication Date


  • 2010

Citation


  • Kulczyk, A. W., Tanner, N. A., Loparo, J. J., Richardson, C. C. & van Oijen, A. M. (2010). Direct observation of enzymes replicating DNA using a single-molecule DNA stretching assay. Journal of Visualized Experiments, 37 e1689-1-e1689-6.

Scopus Eid


  • 2-s2.0-80355140848

Ro Full-text Url


  • http://ro.uow.edu.au/cgi/viewcontent.cgi?article=3205&context=smhpapers

Ro Metadata Url


  • http://ro.uow.edu.au/smhpapers/2187

Start Page


  • e1689-1

End Page


  • e1689-6

Volume


  • 37

Place Of Publication


  • http://www.jove.com/video/1689

Abstract


  • We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. Linearized λ DNA is modified to have a biotin on the end of one strand, and a digoxigenin moiety on the other end of the same strand. The biotinylated end is attached to a functionalized glass coverslip and the digoxigeninated end to a small bead. The assembly of these DNA-bead tethers on the surface of a flow cell allows a laminar flow to be applied to exert a drag force on the bead. As a result, the DNA is stretched close to and parallel to the surface of the coverslip at a force that is determined by the flow rate (Figure 1). The length of the DNA is measured by monitoring the position of the bead. Length differences between single- and double-stranded DNA are utilized to obtain real-time information on the activity of the replication proteins at the fork. Measuring the position of the bead allows precise determination of the rates and processivities of DNA unwinding and polymerization (Figure 2). © JoVE 2006-2011 All Rights Reserved.

Authors


  •   Kulczyk, Arkadiusz W. (external author)
  •   Tanner, Nathan A. (external author)
  •   Loparo, Joseph J. (external author)
  •   Richardson, Charles C. (external author)
  •   van Oijen, Antoine M.

Publication Date


  • 2010

Citation


  • Kulczyk, A. W., Tanner, N. A., Loparo, J. J., Richardson, C. C. & van Oijen, A. M. (2010). Direct observation of enzymes replicating DNA using a single-molecule DNA stretching assay. Journal of Visualized Experiments, 37 e1689-1-e1689-6.

Scopus Eid


  • 2-s2.0-80355140848

Ro Full-text Url


  • http://ro.uow.edu.au/cgi/viewcontent.cgi?article=3205&context=smhpapers

Ro Metadata Url


  • http://ro.uow.edu.au/smhpapers/2187

Start Page


  • e1689-1

End Page


  • e1689-6

Volume


  • 37

Place Of Publication


  • http://www.jove.com/video/1689