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Nucleic Acids Research 2005 33(10):3363-3371; doi:10.1093/nar/gki650
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Published online 9 June 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

Article

A real-time semi-quantitative RT–PCR assay demonstrates that the pilE sequence dictates the frequency and characteristics of pilin antigenic variation in Neisseria gonorrhoeae

Melissa S. Rohrer, Matthew P. Lazio and H. Steven Seifert*

Department of Microbiology-Immunology, Northwestern University Medical School 303 East Chicago Avenue, Chicago, IL 60611, USA

*To whom correspondence should be addressed. Tel: +1 312 503 9788; Fax: +1 312 503 1339; Email: h-seifert{at}northwestern.edu

Received March 1, 2005. Revised May 23, 2005. Accepted May 23, 2005.

A semi-quantitative real-time RT–PCR assay was designed to measure gonococcal pilin antigenicvariation (SQ-PCR Av assay). This assay employs 17 hybridization probe sets that quantitate subpopulations of pilin transcripts carrying different silent pilin copy sequences and one set that detects total pilE transcript levels. Mixtures of a DNA standard carrying the silent copy being detected and a clone encoding the starting pilE sequence, which is the majority pilE template, provided amplification curves that closely matched the experimental data and allowed an analysis of the contribution of different silent pilin copies to variation. The SQ-PCR Av assay was verified using DNA sequence analysis to demonstrate that this methodology allowed an accurate analysis of pilin variation. Both assays showed that with a specific starting pilE sequence, only a subset of the silent pilin copies recombine into pilE at a detectable level, and that this limited subset was reproducibly detected in replicate cultures. When an isogenic pilE sequence variant was examined using both assays, a new subset of silent copy sequences were detected recombining into pilE and the overall frequency of variation was increased. Thus, the parental pilE sequence influences the frequency of variation and the repertoire of pilin variants produced.

Present address: Matthew P. Lazio, University of Iowa, Iowa City, IA 52242, USA


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Home page
 Infect. Immun.Home page
J. K. Hansen, K. P. Demick, J. M. Mansfield, and K. T. Forest
Conserved Regions from Neisseria gonorrhoeae Pilin Are Immunosilent and Not Immunosuppressive
Infect. Immun., August 1, 2007; 75(8): 4138 - 4147.
[Abstract] [Full Text] [PDF]


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