Amici A, Bazzicalupo M, Gallori E, Rollo F
Dipartimento di Biologia Molecolare Cellulare e Animale, Università di Camerino, Italy.
Appl Microbiol Biotechnol. 1991 Nov;36(2):222-7. doi: 10.1007/BF00164424.
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.
为建立一种灵敏可靠的检测方法来监测环境中释放的基因工程微生物（GEMs），通过退火和连接四个合成寡核苷酸构建了一个72bp的双链DNA片段。两个20聚体寡核苷酸的结合位点位于DNA片段内部，位于中心两侧。在构建体的中心部分插入了一个30个核苷酸的识别序列。由于存在两个寡核苷酸结合位点，合成构建体（“车牌”）可以通过聚合酶链反应（PCR）进行酶促扩增，从而使识别系统能够利用该技术出色的灵敏度。当将携带“车牌”染色体的恶臭假单胞菌细胞释放到淡水微观世界中时，只需将煮沸的细胞沉淀物进行PCR扩增，就可以轻松快速地检测到这些细胞。
