Skip Navigation



Nucleic Acids Research Advance Access published online on February 7, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn025
This Article
Right arrow Full Text Freely available
Right arrow Print PDF (5796K) Freely available
Right arrow Screen PDF (271K) Freely available
Right arrowOA All Versions of this Article:
36/4/e24    most recent
gkn025v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Commercial Re-use Guidelines
for Open Access NAR Content
Google Scholar
Right arrow Articles by Osborne, R. J.
Right arrow Articles by Thornton, C. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Osborne, R. J.
Right arrow Articles by Thornton, C. A.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

Cell-free cloning of highly expanded CTG repeats by amplification of dimerized expanded repeats

Robert J. Osborne1,2,* and Charles A. Thornton1

1Department of Neurology, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 14642, USA and 2Department of Physiology, Anatomy and Genetics, University of Oxford, South Parks Road, Oxford, OX1 3QX, UK

*To whom correspondence should be addressed. Tel: +44 1865 282655; Fax: +44 1865 272420; Email: robert.osborne{at}dpag.ox.ac.uk Correspondence may also be addressed to Charles A. Thornton. Tel: +1 585 275 6377; Fax: +1 585 273 1255; Email: charles_thornton{at}urmc.rochester.edu

Received October 26, 2007. Revised January 12, 2008. Accepted January 16, 2008.

We describe conditions for producing uninterrupted expanded CTG repeats consisting of up to 2000 repeats using {phi}29 DNA polymerase. Previously, generation of such repeats was hindered by CTG repeat instability in plasmid vectors maintained in Escherichia coli and poor in vitro ligation of CTG repeat concatemers due to strand slippage. Instead, we used a combination of in vitro ligation and {phi}29 DNA polymerase to amplify DNA. Correctly ligated products generating a dimerized repeat tract formed substrates for rolling circle amplification (RCA). In the presence of two non-complementary primers, hybridizing to either strand of DNA, ligations can be amplified to generate microgram quantities of repeat containing DNA. Additionally, expanded repeats generated by rolling circle amplification can be produced in vectors for expression of expanded CUG (CUGexp) RNA capable of sequestering MBNL1 protein in cell culture. Amplification of dimerized expanded repeats (ADER) opens new possibilities for studies of repeat instability and pathogenesis in myotonic dystrophy, a neurological disorder caused by an expanded CTG repeat.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?




Disclaimer: Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.