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 an...

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Bibliographic Details
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
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Summary: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.
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/15.12.4849