Monitoring a genetically engineered bacterium in a freshwater environment by rapid enzymatic amplification of a synthetic DNA "number-plate".
In order to set up a sensitive and reliable detection method to monitor environmentally released genetically engineered microorganisms (GEMs) a 72-bp, double-stranded DNA fragment has been built by annealing and ligating four synthetic oligonucleotides. Binding sites for two 20-mer oligonucleotides are situated inside the DNA fragment, flanking the centre. Into the central part of the construction a 30-nucleotide identification sequence has been fitted. Thanks to the presence of the two oligonucleotide binding sites, the synthetic construction ("number-plate") can be submitted to enzymatic amplification using the polymerase chain reaction (PCR), thus enabling the identification system to take advantage of the outstanding sensitivity of this technique. When released into a freshwater microcosm, cells of Pseudomonas putida carrying a "number-plated" chromosome could be easily and rapidly detected merely by submitting boiled cell sediments to PCR amplification.
Amici
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Dipartimento di Biologia Molecolare Cellulare e Animale, Università di Camerino, Italy.
Bazzicalupo
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Gallori
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Rollo
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D001483:Base Sequence; D002876:Chromosomes, Bacterial; D004247:DNA / Q000032:analysis* / Q000138:chemical synthesis; D004251:DNA Transposable Elements; D004784:Environmental Monitoring / Q000379:methods*; D005189:False Positive Reactions; D005819:Genetic Markers; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D016133:Polymerase Chain Reaction; D016958:Pseudomonas putida / Q000235:genetics / Q000302:isolation & purification*; D014871:Water Microbiology
D004251:DNA Transposable Elements; D005819:Genetic Markers; D009838:Oligodeoxyribonucleotides; D004247:DNA