Skip Navigation

This Article
Right arrow Full Text Freely available
Right arrow Print PDF (187K) 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 arrowRequest Permissions
Right arrow Commercial Re-use Guidelines
for Open Access NAR Content
Google Scholar
Right arrow Articles by Yang, Y.
Right arrow Articles by Sharan, S. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yang, Y.
Right arrow Articles by Sharan, S. K.
Related Collections
Right arrow Mutagenesis
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Nucleic Acids Research, 2003, Vol. 31, No. 15 e80
© 2003 Oxford University Press

A simple two-step, ‘hit and fix’ method to generate subtle mutations in BACs using short denatured PCR fragments

Yongping Yang and Shyam K. Sharan*

Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute at Frederick, 1050 Boyles Street, Frederick, MD 21702, USA

*To whom correspondence should be addressed at NCI-Frederick, Building 560/Room 32–31C, PO Box B, Frederick, MD 21702, USA. Tel: +1 301 846 5140; Fax: +1 301 846 6323; Email: ssharan{at}mail.ncifcrf.gov

The bacteriophage lambda recombination system has proven to be a valuable tool for engineering bacterial artificial chromosomes (BAC). Due to its high efficiency, subtle alterations in the BACs can be generated using oligonucleotides as targeting vectors. Since no selection marker is used, recombinant clones are identified utilizing a selective PCR screening method. However, occasionally the selective PCR screening is not feasible. We describe here a two-step ‘hit and fix’ method that can be reliably used for generating any subtle alteration in BACs using short denatured PCR fragments as targeting vectors. In the first step of this method, 6–20 nucleotides are changed around the base where the mutation has to be generated. In the second step, these altered nucleotides are reverted to the original sequence and simultaneously a subtle alteration is introduced. Since in each step several nucleotides are changed, PCR primers specific for such alterations can be designed. This two-step method provides a simple and efficient tool for generating subtle alterations in BACs that can be very valuable for functional analysis of genes.


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
 GeneticsHome page
G. L. Bauer, C. Praetorius, K. Bergsteinsdottir, J. H. Hallsson, B. K. Gisladottir, A. Schepsky, D. A. Swing, T. N. O'Sullivan, H. Arnheiter, K. Bismuth, et al.
The Role of MITF Phosphorylation Sites During Coat Color and Eye Development in Mice Analyzed by Bacterial Artificial Chromosome Transgene Rescue
Genetics, October 1, 2009; 183(2): 581 - 594.
[Abstract] [Full Text] [PDF]

Home page
 JCBHome page
J. A. Daniel, M. Pellegrini, J.-H. Lee, T. T. Paull, L. Feigenbaum, and A. Nussenzweig
Multiple autophosphorylation sites are dispensable for murine ATM activation in vivo
J. Cell Biol., December 1, 2008; 183(5): 777 - 783.
[Abstract] [Full Text] [PDF]

Home page
 JEMHome page
S. Difilippantonio, A. Celeste, M. J. Kruhlak, Y. Lee, M. J. Difilippantonio, L. Feigenbaum, S. P. Jackson, P. J. McKinnon, and A. Nussenzweig
Distinct domains in Nbs1 regulate irradiation-induced checkpoints and apoptosis
J. Exp. Med., May 14, 2007; 204(5): 1003 - 1011.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
Q. N. Y. Wong, V. C. W. Ng, M. C. M. Lin, H.-f. Kung, D. Chan, and J.-D. Huang
Efficient and seamless DNA recombineering using a thymidylate synthase A selection system in Escherichia coli
Nucleic Acids Res., March 30, 2005; 33(6): e59 - e59.
[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.