Shabarova Z A, Merenkova I N, Oretskaya T S, Sokolova N I, Skripkin E A, Alexeyeva E V, Balakin A G, Bogdanov A A
Belozersky Laboratory of Molecular Biology and Bioorganic Chemistry, Moscow State University, USSR.
Nucleic Acids Res. 1991 Aug 11;19(15):4247-51. doi: 10.1093/nar/19.15.4247.
An artificial gene comprising 183 base pairs has been assembled by template-directed condensation of 35- to 53-membered oligodeoxyribo nucleotides with cyanogen bromide as a condensing agent. The reaction is complete within several minutes at 0 degrees C in buffer. The resulting mini-gene was cloned and expressed in vivo and in vitro. We have also found that the polymerase inhibition technique (toe-printing) is a good way to ascertain that translation initiation complexes form in the case of single-stranded DNAs as well. Thus, along with the fully chemical assembly of synthetic genes, a rapid and sufficiently reliable method for determining their ribosome-binding properties was developed.
通过以溴化氰作为缩合剂，对35至53个成员的寡聚脱氧核糖核苷酸进行模板导向缩合，组装出了一个由183个碱基对组成的人工基因。该反应在缓冲液中于0摄氏度下几分钟内即可完成。所得的微型基因在体内和体外进行了克隆和表达。我们还发现，聚合酶抑制技术（足迹法）也是确定单链DNA情况下翻译起始复合物是否形成的好方法。因此，除了合成基因的完全化学组装外，还开发了一种快速且足够可靠的方法来确定其核糖体结合特性。
