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DNA Research Advance Access published online on December 17, 2007
DNA Research, doi:10.1093/dnares/dsm023
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© The Author 2007. Kazusa DNA Research Institute
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@oxfordjournals.org

Restriction Site-dependent PCR: An Efficient Technique for Fast Cloning of New Genes of Microorganisms

Yu Jiang1,2, Jianjun Pei1, Xin Song1 and Weilan Shao1,*

1 Jiangsu Key Laboratory for Biodiversity and Bio-Resources, Nanjing Normal University, Nanjing, Jiangsu 210046, People's Republic of China
2 Laboratory of Molecular Microbiology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, People's Republic of China

Received 14 September 2007 ; accepted 14 November 2007.

New bioactive proteins need to be screened from various microorganisms for the increasing need for industrial and pharmaceutical peptide, proteins, or enzymes. A novel polymerase chain reaction (PCR) method, restriction site-dependent PCR (RSD-PCR), was designed for rapid new genes cloning from genomic DNA. RSD-PCR strategy is based on these principles: (i) restriction sites disperse throughout genomes are candidacy for universal pairing; (ii) a universal primer is a combination of a 3'-end of selected restriction sites, and a 5'-end of degenerated sequence. A two-round PCR protocol was designed and optimized for the RSD-PCR: amplify the single strand target template from genomic DNA by a specific primer and amplify the target gene by using the specific primer and one of the universal RSD-primers. The optimized RSD-PCR was successfully applied in chromosome walking using specific internal primers, and cloning of new genes using degenerated primers derived from NH2-terminal amino acid sequence of protein.

Key words: degenerated primer; new gene cloning; polymerase chain reaction

* To whom correspondence should be addressed. Tel. +86 25-85891836. E-mail: wlshao{at}jsmail.com.cn

Edited by Dr Osamu Ohara


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