Solid-phase assembly of cow colostrum trypsin inhibitor gene

• Published in: Nucleic acids research 1987-06, Vol.15 (12), p.4849-4856
• Main Authors: HOSTOMSKY, Z, SMRT, J, ARNOLD, L, TOCIK, Z, PACES, V
• Format: Article
• Language: English
• Subjects: Acrylic Resins; Analytical, structural and metabolic biochemistry; Animals; Base Sequence; Biological and medical sciences; Biotechnology; cattle; Cloning, Molecular; colostrum; Colostrum - analysis; Dna, deoxyribonucleoproteins; Female; Fundamental and applied biological sciences. Psychology; Genes; Genes, Synthetic; Genetic engineering; Genetic technics; Genetic Techniques; Methods. Procedures. Technologies; Nucleic acids; oligonucleotides; Recombinant Proteins - genetics; Synthetic digonucleotides and genes. Sequencing; trypsin inhibitor; Trypsin Inhibitors - genetics
• ISSN: 0305-1048; 1362-4962
• 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.