Hostomský Z, Smrt J, Arnold L, Tocík Z, Paces V
Nucleic Acids Res. 1987 Jun 25;15(12):4849-56. doi: 10.1093/nar/15.12.4849.
A gene for cow colostrum trypsin inhibitor (CTI) was constructed from synthetic oligonucleotides using a novel method of solid-phase gene assembly. In the first step an anchor oligonucleotide was covalently bound to the CNBr-activated Sephacryl S-500 support. Next, triads or tetrads of separately annealed oligonucleotides were stepwise hybridized to the immobilized complementary sequence, with washing after each step. In the last step a linearized vector molecule was ligated to the assembled gene. The whole construct was released from the solid support with a restriction enzyme, circularized, and used for transformation, with a high yield of recombinant clones being obtained. The method represents a generally applicable approach to rapid and efficient assembly of extended DNA duplexes.
采用一种新型的固相基因组装方法，利用合成寡核苷酸构建了牛初乳胰蛋白酶抑制剂（CTI）基因。第一步，将一个锚定寡核苷酸共价连接到经溴化氰活化的Sephacryl S - 500载体上。接下来，将分别退火的寡核苷酸三联体或四联体逐步与固定化的互补序列杂交，每一步之后进行洗涤。最后一步，将线性化的载体分子连接到组装好的基因上。用限制性酶将整个构建体从固相载体上释放出来，环化，并用于转化，获得了高产率的重组克隆。该方法代表了一种普遍适用的快速高效组装延伸DNA双链体的方法。
