Journal Article

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

Yu Jiang, Jianjun Pei, Xin Song and Weilan Shao

in DNA Research

Published on behalf of Kazusa DNA Research Institute

Volume 14, issue 6, pages 283-290
Published in print January 2007 | ISSN: 1340-2838
Published online December 2007 | e-ISSN: 1756-1663 | DOI: http://dx.doi.org/10.1093/dnares/dsm023

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Abstract

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.

Keywords: degenerated primer; new gene cloning; polymerase chain reaction

Journal Article.  5047 words.  Illustrated.

Subjects: Genetics and Genomics

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