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Nucleic Acids Research 2006 34(1):130-139; doi:10.1093/nar/gkj426
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Published online 5 January 2006

© The Author 2006. 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}oxfordjournals.org

Article

Selection of primer-template sequences that bind human immunodeficiency virus reverse transcriptase with high affinity

Jeffrey J. DeStefano* and Jason V. Cristofaro

Department of Cell Biology and Molecular Genetics, University of Maryland Building 231, College Park, MD 20742, USA

*To whom correspondence should be addressed. Tel: +1 301 405 5449; Fax: +1 301 314 9489; Email: jdestefa{at}umd.edu

Received October 26, 2005. Revised November 29, 2005. Accepted December 15, 2005.

A SELEX (systematic evolution of ligands by exponential enrichment)-based approach was developed to determine whether HIV-RT showed preference for particular primer-template sequences. A 70 nt duplex DNA was designed with 20 nt fixed flanking sequences at the 3' and 5' ends and a randomized 30 nt internal sequence. The fixed sequence at the 5' end contained a BbsI site six bases removed from the randomized region. BbsI cuts downstream of its recognition site generating four base 5' overhangs with recessed 3' termini. Cleavage produced a 50 nt template and 46 nt primer with the 3' terminus within the randomized region. HIV-RT was incubated with this substrate and material that bound RT was isolated by gel-shift. The recovered material was treated to regenerate the BbsI site, amplified by PCR, cleaved with BbsI and selected with HIV-RT again. This was repeated for 12 rounds. Material from round 12 bound approximately 10-fold more tightly than starting material. All selected round 12 primer-templates had similar sequence configuration with a 6–8 base G run at the 3' primer terminus, similar to the HIV polypurine tract. Further modifications indicate that the Gs were necessary and sufficient for strong binding.


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