A method for introducing random single point deletions in specific DNA target sequences using oligonucleotides.
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.
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Department of Biochemistry, University of British Columbia, Vancouver, Canada.
Atkinson
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TC
Smith
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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
D004269:DNA, Bacterial; D015345:Oligonucleotide Probes