"Mark the gene": a method for nondestructive introduction of marker sequences inside the gene frame of transgenes.
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.
Morono
Yuki
Y
Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology, Ibaraki, Japan.
Kitagawa
Wataru
W
Kimura
Nobutada
N
Noda
Naohiro
N
Nakamura
Kazunori
K
Kamagata
Yoichi
Y
D000595:Amino Acid Sequence; D001431:Bacteriological Techniques; D003062:Codon / Q000235:genetics; D049308:Dioxygenases / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D018390:Gene Targeting / Q000379:methods*; D005819:Genetic Markers; D005821:Genetic Techniques; D014644:Genetic Variation; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D011995:Recombination, Genetic; D019076:Transgenes
D003062:Codon; D005819:Genetic Markers; D006899:Mixed Function Oxygenases; D049308:Dioxygenases; C074272:2,3-dihydroxybiphenyl oxygenase; C093480:toluene ortho-monooxygenase