Skip Navigation

Nucleic Acids Research 2004 32(21):6187-6199; doi:10.1093/nar/gkh958
This Article
Right arrow Full Text Freely available
Right arrow Print PDF (1299K) 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 ISI Web of Science
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 Search for citing articles in:
ISI Web of Science (6)
Right arrowRequest Permissions
Right arrow Commercial Re-use Guidelines
for Open Access NAR Content
Google Scholar
Right arrow Articles by Chan, S.-h.
Right arrow Articles by Xu, S.-y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chan, S.-h.
Right arrow Articles by Xu, S.-y.
Related Collections
Right arrow Nucleic acid amplification
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Published online 29 November 2004

Nucleic Acids Research, Vol. 32 No. 21 © Oxford University Press 2004; all rights reserved

Cloning of CviPII nicking and modification system from chlorella virus NYs-1 and application of Nt.CviPII in random DNA amplification

Siu-hong Chan, Zhenyu Zhu, James L. Van Etten1 and Shuang-yong Xu*

New England Biolabs, Inc., 32 Tozer Road, Beverly, MA 01915, USA and 1 Department of Plant Pathology and Nebraska Center for Virology, University of Nebraska, Lincoln, NE 68583, USA

* To whom correspondence should be addressed. Tel: +1 978 927 7287; Fax: +1 978 921 1350; Email: xus{at}neb.com

Received October 15, 2004; Revised and Accepted November 5, 2004

The cloning and expression of the CviPII DNA nicking and modification system encoded by chlorella virus NYs-1 is described. The system consists of a co-linear MTase encoding gene (cviPIIM) and a nicking endonuclease encoding gene (cviPIINt) separated by 12 nt. M.CviPII possesses eight conserved amino acid motifs (I to VIII) typical of C5 MTases, but, like another chlorella virus MTase M.CviJI, lacks conserved motifs IX and X. In addition to modification of the first cytosine in CCD (D = A, G or T) sequences, M.CviPII modifies both the first two cytosines in CCAA and CCCG sites as well. Nt.CviPII has significant amino acid sequence similarity to Type II restriction endonuclease CviJI that recognizes an overlapping sequence (RG^CY). Nt.CviPII was expressed in Escherichia coli with or without a His-tag in a host pre-modified by M.CviPII. Recombinant Nt.CviPII recognizes the DNA sequence CCD and cleaves the phosphodiester bond 5' of the first cytosine while the other strand of DNA at this site is not affected. Nt.CviPII displays site preferences with CCR (R = A or G) sites preferred over CCT sites. Nt.CviPII is active from 16 to 65°C with a temperature optimum of 30–45°C. Nt.CviPII can be used to generate single-stranded DNAs (ssDNAs) for isothermal strand-displacement amplification. Nt.CviPII was used in combination with Bst DNA polymerase I large fragment to rapidly amplify anonymous DNA from genomic DNA or from a single bacterial colony.


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
S.-Y. Xu, Z. Zhu, P. Zhang, S.-H. Chan, J. C. Samuelson, J. Xiao, D. Ingalls, and G. G. Wilson
Discovery of natural nicking endonucleases Nb.BsrDI and Nb.BtsI and engineering of top-strand nicking variants from BsrDI and BtsI
Nucleic Acids Res., July 9, 2007; 35(14): 4608 - 4618.
[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.