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Nucleic Acids Research 2005 33(13):e122; doi:10.1093/nar/gni124
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Published online 2 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

SiteFinding-PCR: a simple and efficient PCR method for chromosome walking

Guihong Tan, Yin Gao, Miao Shi, Xinyue Zhang, Shanping He, Zhangliang Chen and Chengcai An*

The National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University Beijing 100871, China

*To whom correspondence should be addressed. Tel: +86 10 62752405; Fax: +86 10 62751526; Email: chcaian{at}pku.edu.cn

Received April 7, 2005. Revised June 27, 2005. Accepted July 18, 2005.

In this paper, we present a novel PCR method, termed SiteFinding-PCR, for gene or chromosome walking. The PCR was primed by a SiteFinder at a low temperature, and then the target molecules were amplified exponentially with gene-specific and SiteFinder primers, and screened out by another gene-specific primer and a vector primer. However, non-target molecules could not be amplified exponentially owing to the suppression effect of stem–loop structure and could not be screened out. This simple method proved to be efficient, reliable, inexpensive and time-saving, and may be suitable for the molecules for which gene-specific primers are available. More importantly, large DNA fragments can be obtained easily using this method. To demonstrate the feasibility and efficiency of SiteFinding-PCR, we employed this method to do chromosome walking and obtained 16 positive results from 17 samples.


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