Construction of a synthetic variant of the bacteriophage f1 gene V by assembling oligodeoxy-nucleotides corresponding to only one strand of DNA |
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Institution: | Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637 U.S.A. |
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Abstract: | A simple and widely applicable procedure for constructing synthetic variants of a gene, involving the synthesis of only one strand of DNA, has been developed. The method is suited for cases in which a cloned DNA with a sequence related to the gene to be constructed is available. First, a heteroduplex DNA which is single-stranded throughout the region of interest is made. This single-stranded region is then used as a template to correctly align and allow ligation of synthetic oligos corresponding to the entire gene. To favor the replication of the strand encoding the synthetic gene, a template strand containing some substitutions of deoxyuridine for deoxythymidine is used. This procedure was used to construct a synthetic bacteriophage f1 gene V which differs from the wild-type (wt) gene at 45 positions out of 298. The synthetic gene was designed to include nine restriction sites without altering the sequence of the encoded DNA-binding protein. The gene construction was found to be very efficient, and about 40 % of the resulting plasmids contained the desired synthetic gene. The synthetic gene was found to be fully active and could substitute for the wt gene in bacteriophage f1. |
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