title: A method for introducing random single point deletions in specific DNA target sequences using oligonucleotides.
abstract: We describe a method for the generation of random point deletions in any target DNA sequence using synthetic mixed oligonucleotides. A mixed pool of oligonucleotides, which contain single nucleotide deletions randomly distributed throughout the full length, was generated by a modification of the synthesis cycle of an automated DNA synthesiser that allowed the inefficient incorporation of nucleotide monomers during each cycle of synthesis. A family of oligonucleotides was used to prime in vitro synthesis of the complementary strand of a cloned DNA fragment in an M13 vector which had previously been passaged through a dut-, ung- Escherichia coli host. Strong selection for progeny from the newly synthesised strand is provided by transforming the heteroduplex into a dut+, ung+ host. This procedure introduced point deletions at 10-25% efficiency. It has been used to introduce point deletions into operator sequences which bind the yeast regulatory proteins encoded by MATa1 and MAT alpha 2.
authors.affiliation: Department of Biochemistry, University of British Columbia, Vancouver, Canada.
authors.forename: S S
authors.initials: SS
authors.lastname: Ner
authors.forename: T C
authors.initials: TC
authors.lastname: Atkinson
authors.forename: M
authors.initials: M
authors.lastname: Smith
mesh_terms: D001483:Base Sequence; D002872:Chromosome Deletion; D003001:Cloning, Molecular / Q000379:methods; D004269:DNA, Bacterial / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D015336:Molecular Probe Techniques / Q000379:methods; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes / Q000138:chemical synthesis; D010957:Plasmids
chemical_list: D004269:DNA, Bacterial; D015345:Oligonucleotide Probes