A method for cloning mixtures of long, synthetic oligodeoxynucleotides.
A method is described for cloning synthetic oligodeoxynucleotides, which can theoretically be of any length. The method requires only a single oligodeoxynucleotide strand and a vector with two unique restriction sites, one of which is for an enzyme that generates 3' protruding ends. A mixture of unpurified oligonucleotides containing a wild-type genetic regulatory sequence of the Escherichia coli gnd gene and two mutations of it was cloned into a plasmid carrying a gnd-lacZ protein fusion. Individual cloned oligonucleotides were readily identified by direct DNA sequencing of plasmid templates. The method is rapid, efficient, and has application to gene synthesis and site-directed mutagenesis.
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D005822:Genetic Vectors; D009154:Mutation; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis*; D010957:Plasmids
D009838:Oligodeoxyribonucleotides