Solid-phase assembly of cow colostrum trypsin inhibitor gene.
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.
Hostomský
Z
Z
Smrt
J
J
Arnold
L
L
Tocík
Z
Z
Paces
V
V
D000180:Acrylic Resins; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003126:Colostrum / Q000032:analysis; D005260:Female; D005796:Genes; D005813:Genes, Synthetic; D005821:Genetic Techniques; D011994:Recombinant Proteins / Q000235:genetics; D014361:Trypsin Inhibitors / Q000235:genetics*
D000180:Acrylic Resins; D011994:Recombinant Proteins; D014361:Trypsin Inhibitors; C053132:sephacryl S 500