In order to set up a sensitive and reliable detection method to monitor environmentally releases 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.

Monitoring a genetically engineered bacterium in a freshwater environment by rapid enzymatic amplification of a synthetic DNA "number plate".

AMICI, Augusto;
1991-01-01

Abstract

In order to set up a sensitive and reliable detection method to monitor environmentally releases 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.
1991
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11581/104170
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