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Nucleic Acids Research 2005 33(14):e126; doi:10.1093/nar/gni111
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Published online 8 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

Multiplex PCR: use of heat-stable Thermus thermophilus RecA protein to minimize non-specific PCR products

Yasushi Shigemori1,2, Tsutomu Mikawa3,4, Takehiko Shibata4 and Michio Oishi1,*

1Kazusa DNA Research Institute 2-6-7 Kazusa-kamatari, Kisarazu, Chiba 292-0818, Japan 2Aisin Cosmos R&D Co., Ltd 2-6-7 Kazusa-kamatari, Kisarazu, Chiba 292-0818, Japan 3RIKEN Harima Institute/SPring-8 Mikazuki cho, Hyogo 679-5148, Japan 4RIKEN Discovery Research Institute 2-1 Hirosawa, Wako, Saitama 351-0198, Japan

*To whom correspondence should be addressed. Tel: +81 438 52 3945; Fax: +81 438 52 3946; Email: oishi{at}kazusa.or.jp

Received May 28, 2005. Revised June 27, 2005. Accepted June 27, 2005.

In this paper we report that the inclusion of heat-resistant RecA protein from a thermophilic bacteria, Thermus thermophilus, and its cofactor (ATP) in PCR effectively eliminates non-specific PCR products. The effect of RecA protein, which catalyzes pairing between homologous DNA molecules with great fidelity in genetic recombination, is due to its promotion of precise priming in PCR (i.e. priming at sites where the primer sequence is completely complementary to that of the target sequence). In addition, the RecA protein substantially reduces the primer concentration required for PCR. These experimental results have led to the realization of multiplex PCR, which involves PCR for multiple sites in the same reaction mixture. We were able to successfully perform multiplex PCR with over a dozen reactions without affecting the amplification pattern of the PCR products.


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