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Nucleic Acids Research 2005 33(15):e130; doi:10.1093/nar/gni129
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Published online 1 September 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oupjournals.org

Methods Online

Rapid generation of long synthetic tandem repeats and its application for analysis in human artificial chromosome formation

Tom Ebersole, Yasuhide Okamoto1, Vladimir N. Noskov, Natalay Kouprina, Jung-Hyun Kim, Sun-Hee Leem, J. Carl Barrett, Hiroshi Masumoto and Vladimir Larionov*

Laboratory of Biosystems and Cancer, National Cancer Institute Bethesda, MD, USA 1Division of Biological Science, Graduate School of Science, Nagoya University Chikusa-ku, Nagoya 464-8602, Japan

*To whom correspondence should be addressed. Tel: +1 301 496 7941; Fax: +1 301 480 2772; Email: larionov{at}mail.nih.gov

Received June 15, 2005. Revised August 3, 2005. Accepted August 3, 2005.

Human artificial chromosomes (HACs) provide a unique opportunity to study kinetochore formation and to develop a new generation of vectors with potential in gene therapy. An investigation into the structural and the functional relationship in centromeric tandem repeats in HACs requires the ability to manipulate repeat substructure efficiently. We describe here a new method to rapidly amplify human alphoid tandem repeats of a few hundred base pairs into long DNA arrays up to 120 kb. The method includes rolling-circle amplification (RCA) of repeats in vitro and assembly of the RCA products by in vivo recombination in yeast. The synthetic arrays are competent in HAC formation when transformed into human cells. As short multimers can be easily modified before amplification, this new technique can identify repeat monomer regions critical for kinetochore seeding. The method may have more general application in elucidating the role of other tandem repeats in chromosome organization and dynamics.


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