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Nucleic Acids Research, 1988, Vol. 16, No. 10 4287-4298
© 1988

Articles

Synthesis of a gene for the HIV transactivator protein TAT by a novel single stranded approach involving in vivo gap repair

Sally E. Adams, Ian D. Johnson, Martin Braddock1, Alan J. Kingsman, Susan M. Kingsman1 and R.Mark Edwards*

Department of Molecular Biology, British Bio-technology Ltd, Brook House Watlington Road, Cowley, Oxford, OX4 5LY 1Department of Biochemistry, University of Oxford South Parks Road, Oxford, OX1 3QU, UK

* To whom correspondence should be addressed

Received March 1, 1988. Revised April 26, 1988. Accepted April 26, 1988.

The synthesis of a gene for the HIV TAT protein is described using a novel approach that capitalises on the ability to synthesise oligonucleotides of greater than 100 bp in length. It involves the synthesis of large oligomers covering one strand of the desired gene in its entirety and the use of small complementary bridging and adapter oligonucleotides to direct the assembly and cloning of the large oligomers. After ligation to the cloning vector the partially single stranded intermediate is transformed directly into the recipient bacterial host where the plasmid is repaired. The synthetic tat gene has been expressed in HeLa cells and is shown to trans-activate TAR+ but not TAR– HIV LTR-CAT constructs.


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