Solid-phase assembly of cow colostrum trypsin inhibitor gene. |
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Authors: | Z Hostomský , J Smrt, L Arnold, Z Tocí k, V Paces |
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Affiliation: | Z Hostomský, J Smrt, L Arnold, Z Tocík, and V Paces |
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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. |
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