Construction of a synthetic variant of the bacteriophage f1 gene V by assembling oligodeoxynucleotides corresponding to only one strand of DNA.
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.
Terwilliger
T C
TC
Department of Biochemistry and Molecular Biology, University of Chicago, IL 60637.
D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics; D005814:Genes, Viral; D005818:Genetic Engineering; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D014764:Viral Proteins / Q000235:genetics
D004274:DNA, Recombinant; D009838:Oligodeoxyribonucleotides; D014764:Viral Proteins