Total synthesis of a gene for bovine rhodopsin. | Zendy

Luca Ferretti | Zendy; Sadashiva S. Karnik | Zendy; H. Gobind Khorana | Zendy; Michael Nassal | Zendy; Daniel D. Oprian | Zendy

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Total synthesis of a gene for bovine rhodopsin.

Author(s) -

Luca Ferretti,

Sadashiva S. Karnik,

H. Gobind Khorana,

Michael Nassal,

Daniel D. Oprian

Publication year - 1986

Publication title -

proceedings of the national academy of sciences

Language(s) - English

Resource type - Journals

SCImago Journal Rank - 5.011

H-Index - 771

eISSN - 1091-6490

pISSN - 0027-8424

DOI - 10.1073/pnas.83.3.599

Subject(s) - oligonucleotide , gene , biology , nucleotide , restriction site , restriction enzyme , dna , base pair , mutagenesis , genetics , rhodopsin , restriction map , site directed mutagenesis , microbiology and biotechnology , nucleic acid sequence , biochemistry , mutant , retinal

To carry out systematic structure-function studies of bovine rhodopsin by specific amino acid replacements, we have accomplished the total synthesis of its gene, which is 1057 base pairs long. The synthetic gene contains 28 unique restriction sites that are on the average 60 base pairs apart. Replacement of specific restriction fragments by synthetic counterparts containing the desired nucleotide changes permits specific mutagenesis in all parts of the gene. The synthesis of the gene involved enzymatic joining of a total of 72 synthetic oligonucleotides, 15-40 nucleotides long, to form DNA duplexes. The total gene was assembled from three synthetic fragments that were cloned. All synthetic oligonucleotides were characterized by 5'-end analysis, and the accuracy of the joining reactions was confirmed by sequencing the three fragments as well as the complete gene.

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