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"Mark the Gene": a Method for Nondestructive Introduction of Marker Sequences Inside the Gene Frame of Transgenes ^,

Yuki Morono,^1, Wataru Kitagawa,^2* Nobutada Kimura,¹ Naohiro Noda,¹ Kazunori Nakamura,¹ and Yoichi Kamagata^1,2

Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan,¹ Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology, 2-17-2-1, Tsukisamu-Higashi, Toyohira, Sapporo 062-8517, Japan²

Received 11 January 2007/ Accepted 20 May 2007

A specific marking and detection technique is a fundamental requirement for the safer use of genetically modified (GM) organisms. Here we propose a simple and effective method for directly marking functional transgenes in GM organisms. For that purpose, we introduced nucleotide substitutions (NS), based on the degeneracy of codons as markers (NS markers), into the bphC (2,3-dihydroxybiphenyl dioxygenase) and tomA3 (toluene-ortho-monooxygenase) gene frames using a PCR-based method. No change was observed in the enzyme activity of translated proteins, and alignments with homologous genes showed the uniqueness of the NS markers. Furthermore, we constructed tomA3 variations harboring NS markers in different positions. Although the translational products were identical, the constructed variation genes could be distinguished through their marker patterns by multiplex PCR, showing that NS markers could serve as product-specific tags for identifying individual GM organisms. This direct method of marking the functional transgene provides a simple, low-risk, and robust marking method without causing the gene functions to deteriorate.

Published ahead of print on 25 May 2007.

Supplemental material for this article may be found at http://aem.asm.org/.

Present address: Kochi Institute for Core Sample Research, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Monobe B200, Nankoku, Kochi 783-8502, Japan.