Solid-phase assembly of cow colostrum trypsin inhibitor gene
- 1 January 1987
- journal article
- Published by Oxford University Press (OUP) in Nucleic Acids Research
- Vol. 15 (12) , 4849-4856
- https://doi.org/10.1093/nar/15.12.4849
Abstract
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.Keywords
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