Skip Navigation

Nucleic Acids Research 2005 33(13):e117; doi:10.1093/nar/gni116
This Article
Right arrow Full Text Freely available
Right arrow Print PDF (896K) Freely available
Right arrow Screen PDF (252K) Freely available
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 Müller, K. M.
Right arrow Articles by Arndt, K. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Müller, K. M.
Right arrow Articles by Arndt, K. M.
Related Collections
Right arrow Mutagenesis
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Published online 1 August 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

Nucleotide exchange and excision technology (NExT) DNA shuffling: a robust method for DNA fragmentation and directed evolution

Kristian M. Müller*, Sabine C. Stebel, Susanne Knall, Gregor Zipf1, Hubert S. Bernauer1 and Katja M. Arndt

Institut für Biologie III, Universität Freiburg Schänzlestraße 1, 79104 Freiburg, Germany 1ATG:Biosynthetics Freiburg, Germany

*To whom correspondence should be addressed. Tel: +49 761 203 2748; Fax: +49 761 203 2745; Email: kristian{at}biologie.uni-freiburg.de

Received March 30, 2005. Revised July 8, 2005. Accepted July 8, 2005.

DNA shuffling is widely used for optimizing complex properties contained within DNA and proteins. Demonstrated here is the amplification of a gene library by PCR using uridine triphosphate (dUTP) as a fragmentation defining exchange nucleotide with thymidine, together with the three other nucleotides. The incorporated uracil bases were excised using uracil-DNA-glycosylase and the DNA backbone subsequently cleaved with piperidine. These end-point reactions required no adjustments. Polyacrylamide urea gels demonstrated adjustable fragmentation size over a wide range. The oligonucleotide pool was reassembled by internal primer extension to full length with a proofreading polymerase to improve yield over Taq. We present a computer program that accurately predicts the fragmentation pattern and yields all possible fragment sequences with their respective likelihood of occurrence, taking the guesswork out of the fragmentation. The technique has been demonstrated by shuffling chloramphenicol acetyltransferase gene libraries. A 33% dUTP PCR resulted in shuffled clones with an average parental fragment size of 86 bases even without employment of a fragment size separation, and revealed a low mutation rate (0.1%). NExT DNA fragmentation is rational, easily executed and reproducible, making it superior to other techniques. Additionally, NExT could feasibly be applied to several other nucleotide analogs.


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


This article has been cited by other articles:


Home page
 Nucleic Acids ResHome page
L. Liu, Y. Li, D. Liotta, and S. Lutz
Directed evolution of an orthogonal nucleoside analog kinase via fluorescence-activated cell sorting
Nucleic Acids Res., July 1, 2009; 37(13): 4472 - 4481.
[Abstract] [Full Text] [PDF]

Home page
 Appl. Environ. Microbiol.Home page
W.-T. Liu, H. Guo, and J.-H. Wu
Effects of Target Length on the Hybridization Efficiency and Specificity of rRNA-Based Oligonucleotide Microarrays
Appl. Envir. Microbiol., January 1, 2007; 73(1): 73 - 82.
[Abstract] [Full Text] [PDF]


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.